Review
Experimental models of chronic subdural hematoma Josephine A. D’Abbondanza1,2, R. Loch Macdonald1,2 1
Division of Neurosurgery, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario, Canada, 2 Department of Surgery, Institute of Medical Science, University of Toronto, Ontario, Canada Objectives: Chronic subdural hematoma (CSDH) is a common neurosurgical problem. Most studies of pathogenesis and treatment involve humans. Advances in understanding of human diseases may be made using animal models. We reviewed all animal models of CSDH and report here their results, conclusions and limitations in order to set a baseline upon which further advanced experimental work related to this disease can be made. Methods: PubMed, Medline, Embase and ISI Web of Knowledge were searched with no time limits using the keyword ‘chronic subdural hematoma’ and MeSH term ‘hematoma, subdural, chronic’. The authors reviewed all papers written related to this disease and selected all publications involving animals. There were no other restrictions. The findings and conclusions of the papers are summarized here. No formal analysis was done because of the variation in species used, methods for induction of CSDH, times of assessment and reporting of results. Results: Attempts to create CSDH have been made in mice, rats, cats, dogs and monkeys. Methods include injection or surgical implantation of clotted blood or various other blood products and mixtures into the potential subdural space or the subcutaneous space. No intracranial model produced a progressively expanding CSDH. Transient hematoma expansion with liquification could be produced by subcutaneous injections in some models. Spontaneous subdural blood collections were found after creation of hydrocephalus in mice by systemic injection of the neurotoxin, 6-aminonicotinamide. The histology of the hematoma membranes in several models resembles the appearance in humans. None of the models has been replicated since its first description. Discussion: We did not find a report of a reproducible, well-described animal model of human CSDH. Keywords: Chronic subdural hematoma, Experimental model
Introduction Chronic subdural hematoma (CSDH) is a form of hematoma occurring between the dura and arachnoid mater. It is hypothesized to be caused by traumatic brain injury, where shearing forces cause bridging veins to tear where they traverse the potential subdural space and enter the dura.1 Chronic subdural hematoma is more common with an increase in age. Other factors associated with development of CSDH are chronic alcohol abuse, ventriculoperitoneal shunting and other factors associated with cerebral atrophy. Chronic subdural hematoma generally develops a few days to weeks after minor trauma, but these incidents are not documented in up to 50% of the patients. The clinical presentation can be acute,
Correspondence to: Josephine A. D’Abbondanza, Division of Neurosurgery, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 209 Victoria Street, Toronto, Ontario M5B 1T8, Canada. Email: [email protected]
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ß W. S. Maney & Son Ltd 2014 DOI 10.1179/1743132813Y.0000000279
however, it is usually secondary to acute bleeding into the CSDH. Chronic subdural hematoma differs from acute subdural hematoma (ASDH) in terms of the speed of onset, as ASDH develops much faster after head injury.1 Bleeding in both ASDH and CSDH is usually venous and subsequently slower than arterial epidural hemorrhage.1 Chronic subdural hematoma is common in neurosurgical practice and it is understudied relative to its prevalence. Two studies estimated the incidence. Fogelholm and Waltimo reported that the incidence of CSDH in Helsinki between 1967 and 1973 was 1.7 per 100 000 per year, with a steep rise in incidence with an increase in age.2 Kudo et al. found an overall incidence of 13 per 100 000 per year in Awaji Island, Japan, based on the data collected between 1986 and 1988.3 Again there was a marked increase with an increase in age. The mean age of those affected with CSDH is not well documented other than in multiple, mostly retrospective, case series where the
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mean age ranged from 50 to 67 in a review of eight case series4 and about 75-years-old in a study from the United Kingdom.5 Males constitute about 75% of cases.5 Given that the proportion of people over 65-years-old is predicted to double worldwide between 2000 and 2030, CSDH will become an even more frequent health care problem.6 The expanding indications for anticoagulation in the elderly also may increase the incidence.7 While the treatment is relatively simple, the disease is associated with substantial morbidity, including recurrence rates of 5–30%.8 Mortality 6 months after treatment was 13% (28 of 210 patients) in a clinical trial that accrued patients between 2004 and 20075 and 26% (55 of 209 patients) in a series of cases collected since 2000.9 Although many studies have focused on clinical treatment and a few on pathogenesis and pathology in humans, there has been less attention to experimental models and investigation of CSDH. While the study of humans is of great interest, an animal model could lead to pathophysiologic insights and perhaps new treatments. The purpose of this paper is to review animal models of CSDH. We did not find guidelines for systematic reviews of animal models of diseases but we began by following the PRISMA structure.10 The rationale for this review is to summarize what has been previously attempted for the experimental study of CSDH, so that those interested in developing CSDH models or in studying CSDH have a baseline upon which they can move forward. The objective of this article was to summarize every study attempting to or actually creating an experimental model of CSDH.
Methods PubMed, Medline, Embase and ISI Web of Knowledge were searched in September 2013 with no date limits. The keyword was ‘chronic subdural hematoma’. The MeSH term ‘hematoma, subdural, chronic’ was also included. The titles, abstracts and key words of every paper related to this disease were searched and then papers were selected from this search and from the reference lists of the reviewed papers. There was no review protocol. The only eligibility criterion was that animals were included in the paper. There was no language restriction. The two authors performed the above process individually and then collected the data on the species used, methods, results and conclusions of the included papers. Both authors summarized the data: the first author wrote the narrative description and the second author revised it. No formal analysis was done because of the variation in species used, methods for induction of CSDH, times of assessment,
Experimental Models of Chronic Subdural Hematoma
lack of quantitative data and variable reporting of results.
Results The initial search showed 2997 references. The titles and abstracts were reviewed and five publications were found that used animals to study CSDH. None of the remaining reports was an animal study. The reference lists of the five papers were searched, which disclosed an additional eight papers. Finally, a selection of the 2997 references (241) were read and the reference lists reviewed to see if there are any other studies involving animals. This found no additional papers. There are a number of papers found that were published more than 85 years ago that did not provide any useful information; in these papers the species, surgical procedure or results were not mentioned or lacked sufficient details for analysis. They are summarized for historical interest because they show that some current understanding, whether correct or not, originated from those papers. The more recent included studies are in the tables (Tables 1–4).
Early models Putnam and Putnam reviewed studies attempting to produce CSDH in animals.11 Serres tore the longitudinal sinus subdurally in animals and noted that the animals developed seizures and hemiplegia within hours.12 When the animals were euthanized within a few days, there was liquid blood in a fibrinous sac that was adherent to the dura. These observations within days of hemorrhage would not represent CSDH. Similarly, Laborde injected subdural blood in dogs and cats but sacrificed them after a day.13 Sperling and others created trephines in the skulls of rabbits and injected whole blood under the dura.14,15 When animals were sacrificed after 4 days to 6 weeks, a thin, brown, vascular membrane was almost always found on the inner surface of the dura, likely representing organization of the hematoma. In humans, CSDH sometimes has a loculated appearance. Inflammation of the dura, theoretically initiated by blood and fibrin exposure, results in the formation of a neomembrane around a liquified clot.1 Other processes postulated to contribute to the pathogenesis of CSDH are fibrinolytic activity causing repeated hemorrhage into the CSDH. Sperling does not describe a liquified hematoma and he provided no illustrations of the membrane; therefore, it is difficult to infer similarities to human CSDH solely by his descriptions. He concluded that CSDH was produced by capillary or venous hemorrhage into the subdural space. This was in contrast to the prevailing theory at that time, which states that CSDH was an inflammatory lesion, possibly due to infection.16
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1944
1963
Gardner
Christensen
Goodell and Mealey
1932
1927
Putnam and Putnam
Gardner
Year
Author
Dog (n 5 9)
Dog (n 5 2–4 per group)
Dog (n 5 7)
Dog (n 5 5)
Dog (n 5 3), cat (n 5 15)
Species
Subdural injection, with or without urea to reduce brain volume and allow injection of more blood
Subdural injection with or without ligation of superior sagittal sinus, ligation of superior sagittal sinus alone
Subdural injection to the contralateral subdural space with a curved needle through a 1–2 cm trephine
Subdural injection through a 1–2 cm trephine
Subdural injection through a 1–2 cm trephine
Method of injection
3.8% citrate blood or paraffin (to prevent clotting) blood, also tried repeated blood injections and blood injection plus head injury, volume not stated
Autologous fresh blood
Subdural space
Autologous unclotted whole blood
Subdural space
Subdural space
Autologous unclotted whole blood
Autologous whole blood, defibrinated blood, washed fibrin
Substances injected/volume
Subdural space
Subdural space
Site of injection
Table 1 Blood injection models of chronic subdural hematoma (CSDH)
2–12 ml once, repeated injections weekly of up to 27 ml
Not stated
3– 11.5 ml
0.7–3 ml
1–2 ml
Volumes
Up to 84 days
8 days to 5 weeks
Not stated
3–14 weeks
1 day to 3 months
Duration of study
Subdural hematomas that organized and resolved over time
No gross evidence of injected blood if euthanasia was more than couple of weeks after injection Residual organizing hematoma with membranes that resolved with time
No gross evidence of injected blood
Small remaining volumes of subdural blood, occasional membranes
Gross pathology
No evidence of subdural hematoma in most animals, some showed a fibrous subdural membrane
Outer membrane of fibroblasts, capillaries, large vascular channels, organizing hematoma with no liquidcontaining cavity
Not mentioned
Not mentioned
Outer membrane of granulation tissue with neovascularity, organizing hematoma, thin inner mesothelial membrane
Microscopy
Chronic subdural hematoma is of traumatic origin, ligating the superior sagittal sinus slowed clearance of subdural blood, this model was similar to human CSDH except that the small volumes of blood injected completely organized rather than liquifying Subdural blood alone was insufficient to produce a CSDH, the osmotic theory is untenable
Pachymeningitis could be produced by injection of blood or fibrin or by chronic alcohol intake, but progressive lesions like in humans could not be created It is difficult if not impossible to reproduce a CSDH in dogs, ascribed to the osmotic theory of hematoma enlargement It is difficult if not impossible to reproduce a CSDH in dogs, ascribed to the osmotic theory of hematoma enlargement
Conclusions
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2004
Eijkenboom
2008
1982
Ohshima
Jussen
Year
Continued
Author
Table 1
Rat (n 5 56)
Rat (n 5 115 in five groups)
Dog (n 5 45)
Species
Subdural space
Subdural space
Subdural injection after craniotomy
Subdural injection after craniotomy
Subdural space
Site of injection
Subdural injection after inflating a balloon in the subdural space to create a cavity
Method of injection
Autologous fresh blood
Autologous fresh blood
Autologous clotted blood, fresh blood, blood mixed with cerebrospinal fluid, blood mixed with cerebrospinal fluid plus systemic mannitol and heparin
Substances injected/volume
4 ml
1 ml bilaterally
3–4 ml
Volumes
Up to 12 days
1–18 weeks
Up to 21 days
Duration of study
Clot removed after 60 minutes and local cerebral blood flow was similar across all groups
Computed tomography (CT) showed resolving hematomas if only fresh or clotted blood was injected, mixing with cerebrospinal fluid (CSF) lowers the density of the hematoma on CT but it still resolved, 3 of 10 dogs treated with blood plus heparin plus mannitol showed expanding hematomas until euthanasia in 21 days Hematomas observed in the sensorimotor cortex and other lesions, including ventricle dilations and compression damage
Gross pathology
Calcium deposits in thalamic nuclei, astrocyte reactivity evident early followed by the disappearance at week 8 and strong reappearance at week 18 Contralateral infarction and neurological scores due to hematoma were reduced with treatment
The outer membrane of granulation tissue, capillaries, expansion was due to hemorrhage from the outer membrane neovascularity
Microscopy
Hypertonic/hyperoncotic treatment (HHT) and surgical evacuation reduced intracranial pressure improved functional outcome
Bilateral subdural hematoma caused impairment in spatial navigation that recovered by week 8 but reoccurred at week 18
A combination of blood plus cerebrospinal fluid plus recurrent hemorrhage from the outer membrane needed to form a chronic expanding hematoma
Conclusions
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1974 Dog Subdural or Subdural or (n 5 2) and subcutaneous subcutaneous cat (n 5 40) injection, space polyethylene sheet between dura and hematoma and devascularization of dura by excision and reinsertion
Apfelbaum et al.
Subdural or subcutaneous space
Subdural space
Site of injection
1972 Dog (n 5 62), monkey (n 5 5)
Subdural injection, with or without urea to reduce the brain volume and allow injection of more blood, some animals had cisternopleural shunts Subdural injection after inflating a balloon in the subdural space to create a cavity, or subcutaneous injection
Method of injection
Watanabe et al.
Goodell 1963 Dog and Mealey (n 5 52 in six groups)
Author
NO .
2
Autologous blood with or without cerebrospinal fluid, defibrinated blood
Autologous clotted blood with or without cerebrospinal fluid
Autologous clot, with or without cerebrospinal fluid, streptokinase, or hydrocortisone, frozen and thawed blood
Substances injected/volume
Table 2 Clot injection and placement models of chronic subdural hematoma (CSDH) Duration of study
1–5 ml
8–30 days
7–14 days in dogs, 14–21 days in monkeys
Up to 2–12 ml once, 84 days repeated injections weekly of up to 27 ml
Volumes
Subdural hematomas that organized and resolved over time
Three dogs developed enlarging subdural fluid collections, the rest showed resolving hematomas
Subdural hematomas that organized and resolved over time
Gross pathology
The outer membrane with sinusoidal vascular channels lined by a single layer of endothelial cells, loose connective tissue, macrophages, fibroblasts and inflammatory cells, thin inner membrane. Similar histology in the subcutaneous space with some dogs developing expanding hematomas, removing fibrin from the clots with plasmin or implanting fibrin plus thrombin did not cause an expanding hematoma, only expanding hematomas were with blood containing fibrin plus cerebrospinal fluid Outer membranes with fibroblasts, neovascularity, extravasation of erythrocytes was observed from the neovascularity, thin inner membrane, liquifying hematoma
The outer membrane with neovascularity, fibroblasts, phagocytes, organizing hematoma and thin, single cell layer inner membrane, injection of other substances with the blood did not affect the findings
Microscopy
Cerebrospinal fluid is not necessary for CSDH formation but fibrin is, the histologic response to extravascular blood clot is the same in the subdural space as in other body spaces, CSDH expansion is due to bleeding from the neovascularity in the membranes
A combination of blood containing fibrin plus cerebrospinal fluid is necessary to produce an expanding fluid collection
Subdural blood alone was insufficient to produce a CSDH, the osmotic theory is untenable
Conclusions
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1976 Rat (n 5 154)
Labadie and Glover
Subcutaneous implantation or injection
Method of injection
Substances injected/volume
Subcutaneous Human plasma, space plasma plus cerebrospinal fluid, plasma plus carrageenan, autologous hemolyzed blood, autologous fresh blood
Site of injection
Year
1963
Author
Goodell and Mealey
Dog (n 5 7)
Species
Avulsion of bridging veins
Method of injection No blood injected
Site of injection
Volumes 9 days
No blood injected
Microscopy
Up to 72 days
Subdural hematomas that organized and resolved over time
Gross pathology
Surviving animals showed reabsorption or no evidence of hematoma/fibrous membrane
Microscopy
Most hematomas Fibroblastic resolved, hematomas membrane with that expanded with neovascularity liquified contents between about 10 and 20 days were larger volumes of plasma alone if surgically implanted (43%) but not if injected (0%), plasma plus carrageenan (33–50%), autologous hemolyzed or whole blood 45–50%)
Gross pathology
Duration of study
Duration of study
Volumes
2–16 ml
Tearing of bridging veins using silk sutures, intravenous hypertonic urea or heparin
Substances injected/volume
Table 3 Vessel avulsion models of chronic subdural hematoma (CSDH)
Year
Author
Species
Continued
Table 2
Subdural blood from torn bridging veins was insufficient to produce a CSDH, the bridging vein theory is untenable
Conclusions
The composition and volume of clot are important for developing an expanding hematoma, plasma–fibrin provides the matrix shape for the hematoma, membrane formation requires erythrocytes, hemoglobin, leucocytes, other blood elements and inflammation whereas cerebrospinal fluid is not necessary
Conclusions
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Hematomas formed when the ventricular system spontaneously decompressed into the subdural space, collapsing the cortical mantle and tearing bridging veins Hematomas surrounded by a thin inner membrane and a thick outer membrane with fibroblasts, neovascularity, hemosiderin-laden macrophages and fresh hemorrhage No blood injected
Creation of hydrocephalus in neonatal mice by intraperitoneal injection of 6aminonicotinamide
No blood injected
Up to 30 days
Hydrocephalus, 60% developed spontaneous subdural hematomas
Putnam and Putnam created 1–2 cm trephines at the junction of the longitudinal and transverse sinuses in dogs and cats and injected autologous blood into the subdural space.11 Sometimes the blood was inadvertently injected into the subarachnoid space. The blood also had a tendency to leak out through the needle hole. In an attempt to correct this, washed fibrin was injected into some animals. The animals were sacrificed after 1–50 days. In some cases it was possible to produce a thin neomembrane adherent to the dura that overlay a hematoma. However, the border between the membrane and clot was not as distinct as in human CSDH and there was no liquified hematoma cavity. Empty mesothelial-lined spaces, similar to those in human outer CSDH membranes, were found throughout the membrane and were sometimes found lining the dura. Gardner subjected five dogs to subdural injection of autologous unclotted blood and euthanasia after 3–14 weeks.19 Little or no blood was visible beneath the dura of these animals. He also made a burr hole on one side of the head in seven dogs and inserted a curved needle through the hole and across the midline to the contralateral convexity subdural space and injected 3–11.5 ml unclotted autologous blood. When animals were euthanized more than a couple of weeks after there were no hematomas remaining. Additionally, Gardner placed cellophane sacs containing autologous whole blood into the subdural space of eight dogs. The sacs were removed after 3–18 days and were found to be 39–103% heavier. This was taken as evidence that an osmotic gradient (high osmolarity in the hematoma compared to the cerebrospinal fluid [CSF]) caused the hematoma to imbibe fluid and create a CSDH. Christensen performed subdural injections of citrated blood in dogs by sacrificing them after 8–11 days.20 Blood was injected every week for 3 weeks in two dogs and the animals were euthanized
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Other investigators injected a mixture of fatty acids into the subdural space and produced dural thickening and occasionally a laminated hemorrhage.11 None of these studies was accompanied by histology. Physicians in the nineteenth century recognized that CDSH or pachymeningitis hemorrhagica interna, as it was called, was more common in alcoholics. Kremiansky conducted experiments on dogs, feeding them brandy every day for 2 or 3 months. He found a pachymeningitis in 3 out of 4 experiments and concluded that alcohol caused inflammation that caused the subdural lesions.17 His experiments on dogs have been repeated by a number of investigators, and subdural membranes have occasionally, although usually not, been produced in animals.18
Blood injection models
Mouse (n 5 38)
Substances injected/volume Site of injection Year
Species
Method of injection
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Table 4 Spontaneous hematoma models of chronic subdural hematoma (CSDH)
Volumes
Duration of study
Gross pathology
Microscopy
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1 week after the last injection. How the injections were performed is not stated. There was proliferation of blood vessels and fibrous tissue forming a structure similar to the outer membrane of a CSDH. The thick outer membrane produced consisted of fibroblasts, capillaries up to 50 mm in diameter (similar to the large sinusoidal vessels seen in human outer CSDH membranes) and other cells. Deep to this was some residual blood with decaying erythrocytes and infiltrating macrophages but no liquified hematoma cavity. In this investigator experiments, injecting subdural blood and then inflicting head trauma weekly for 3 weeks also did not produce a CSDH. Finally, in four dogs, the superior sagittal sinus was ligated and subdural paraffin (to reduce clotting) blood injected bilaterally into the subdural space. After 11 days to 3 weeks, during euthanasia, histology showed some larger cavities in the subdural space, in addition to the typical outer membrane with neovascularization and fibroblasts. He concluded that injection of subdural blood along with ligation of the superior sagittal sinus in dogs produced a lesion similar to CSDH. In 1963, Goodell and Mealey produced CSDH by injecting 2–10 ml autologous blood subdurally through a burr hole.21 A needle and syringe or a polyethylene catheter was sealed in the subdural space so that blood injected did not leak out of the dural opening into the epidural space. This was a problem with previous needle injection methods. Intravenous urea was usually given in order to reduce the intracranial pressure and permit injection of larger blood volumes. Of the eight dogs with subdural blood injections sacrificed after 8–72 days, 1 died acutely, 5 showed no evidence of hematoma formation and 2 had thin residual membrane or dural thickening. Multiple subdural injections (2–5) of whole blood were administered over 12–35 days to eight dogs. Five survived and exhibited the same pattern of organization and resolution of the blood as seen after single injections, without formation of a CSDH. The investigators recognized the role of craniocerebral disproportion in producing a CSDH and attempted to induce this by injecting subdural blood in dogs with cisternopleural shunts or by administering urea to the dogs for days after the injections. No change in the histology of the hematoma resolution occurred. Subdural injection of blood mixed with CSF, streptokinase, cortisone or frozen and then thawed blood also failed to produce a CSDH. They concluded that subdural blood alone was insufficient to produce a CSDH. They considered the osmotic mechanism of CSDH expansion to be untenable. Ohshima injected fresh autologous blood and blood mixed with CSF into the subdural space of
Experimental Models of Chronic Subdural Hematoma
dogs.22 The blood injections followed the method of Watanabe et al., where a balloon was used to expand the subdural space before the injection.23 In the group injected with blood mixed with CSF, some animals were treated with daily doses of mannitol and heparin. They were imaged with computed tomography (CT) and then examined histologically at various times. All of the hematomas slowly resolved except for 3 of 10 in dogs treated with blood plus CSF, mannitol and heparin, in which the hematomas enlarged over time. Histological examination showed typical outer neovascular, fibrous, thickened membranes that were more marked in dogs injected with blood mixed with CSF. The neomembranes were structurally similar to those seen in human CSDH but were not as expansive. The few animals treated with mannitol and heparin that had expanding hematomas showed evidence of hemorrhage from the neovascular outer membrane. The continuing expansion of blood plus CSF reported by Watanabe et al. was not replicated by Ohshima. Thus, the author concluded that CSF mixing with the blood and hemorrhage from the outer membrane were important in the pathogenesis of CSDH. Eijkenboom et al. induced bilateral subdural hematomas above the somatosensory cortex in rats24 by modifying an ASDH model established by Miller et al. in 1990.25 Briefly, a craniotomy was performed and autologous, non-heparinized venous blood was injected into the subdural space. Hematoma placement was verified 1–2 days after surgery by neurological examination of the animals, and the Morris water maze task was subsequently used to test spatial navigation. Histologically, ventricular dilation and damage to the brain secondary to compression were observed. Impairment in spatial navigation was seen but recovered completely by 8 weeks after surgery. However, 18 weeks after surgery further deterioration was evident and resulted in impaired water maze performance. These contrasting findings at week 8 and week 18 may be an interesting target for future studies. In another study of ASDH by Jussen et al., the chronic effects of hypertonic/hyperoncotic treatment (HHT) and surgical evacuation were also assessed, which may be relevant to studies of CSDH.26 The previously described model by Miller et al. was also used, but for the chronic study, fluids were infused after 30 minutes and subdural blood was evacuated 60 minutes after ASDH. Neurological deficits were assessed on 1–11 days after ASDH and histology was performed on day 12. Arterial blood gases remained constant for the duration of the study. Functional outcome improved over time, but on day 11, the neurological state improved and the infarction size was reduced with HHT alone or when it was
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combined with clot evacuation; there was no additional benefit to the combination treatment.
Clot injection and placement models Subdural clots In 21 dogs, Goodell and Mealey injected clotted autologous blood, from which the serum was removed, into the subdural space via a burr hole.21 Clots were introduced 2–5 times over 12–35 days. Urea and lumbar CSF drainage were used to allow injection of up to 12 ml clot. Pathologic examination over time showed formation of an outer membrane composed of fibroblasts and cells phagocytosing the erythrocytes, a layer of decaying erythrocytes, and an inner membrane that is one cell layer thick, thus resembling the membranes of a CSDH. By 4 weeks there were outer and inner membranes surrounding a collection of proteinaceous fluid and decaying erythrocytes. After 4 weeks, the hematomas resolved. Watanabe et al. tested the effects of subdural injections of autologous blood mixed with CSF incubated ex vivo for 24 hours before implantation.23 Subdural inoculation was performed in 12 dogs by inserting a polyethylene tube connected to a balloon into the subdural space through a 3 cm trephine opening. The balloon was filled slowly, evacuated and then blood plus CSF were injected into the preformed cavity. Three animals developed progressive neurological deficits and were euthanized 7–14 days after the injections. They had loculated, liquified hematomas ranging from 6–10 ml in volume. This was more than the 1–5 ml that was injected initially, suggesting that some hematoma expansion had occurred. In nine dogs, there were no neurological symptoms and the volumes found at euthanasia after 7–14 days were similar to what was injected. The outer membranes in all cases were similar to those seen in humans with numerous sinusoidal, dilated vascular channels lined by a single layer of endothelial cells, loose connective tissue, fibroblasts, macrophages and inflammatory cells. A thin inner membrane was also seen. Watanabe et al. also injected 3–4 ml autologous blood mixed with CSF into the subdural space of 5 monkeys using the same method they used in dogs.23 Three monkeys were sacrificed 14 days and 2 monkeys were sacrificed 21 days after injection. No animals developed neurological symptoms or signs, but one animal euthanized after 21 days had a liquified hematoma much larger than the original injected volume. All hematomas were well encapsulated and histologically similar to those found in the dogs and in humans. Apfelbaum et al. noted the work of Watanabe et al. that suggested that CSF was important in the pathogenesis of CSDH based on the theory that it was drawn osmotically into the hematoma across the inner
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membrane.27 They noted that measurements of the osmolarity of the hematoma fluid in humans showed that the osmotic theory was incorrect.28 In an attempt to replicate the findings of Watanabe et al., Apfelbaum et al. conducted a similar experiment in 2 dogs.27 Clotted blood with or without CSF was implanted by the same method into the subdural space. They were sacrificed on the 13th day following clot injections. The subdural hematomas showed neovascular outer and thin inner membranes with some evidence of fresh bleeding into the hematoma from the outer membrane. CSF had no effect on subdural clot absorption and clots did not reach previously reported sizes.23 Additionally, Apfelbaum et al. created a subdural cavity in cats using the method of Watanabe et al.23 Clots consisting of autologous blood or blood mixed with autologous CSF, artificial CSF or saline were incubated at 37uC for 24 hours ex vivo. They were implanted in the subdural space and animals euthanized at varying times up to 30 days. To identify the factors involved in membrane formation, 10 cats were given additional injections of human or bovine fibrinogen. Four cats also had clots isolated from the dura by a sheet of polyethylene and in another group, a disc of dura was cut out to devascularize it, and then resutured in place and the subdural pocket was created below it. All of the injections produced the same pattern of histologic changes with formation of a thick vascular outer membrane and a thin inner membrane. The outer membrane did not form if the clot was isolated from the dura by polyethylene and was delayed in appearance and thinner if the dura had been devascularized. The authors observed extravasation into the decaying hematoma of intact fresh erythrocytes from the thin-walled sinusoidal channels in the outer membrane. The presence of CSF had no effect. All clots disappeared after 21 days and left behind thickened dura. These findings suggest that CSF is not necessary for membrane formation or clot development. Fibrin seemed essential for the formation of the outer membrane because the outer membrane formed if fibrinogen was implanted subdurally but not if defibrinated blood was. The authors concluded that encapsulation of the hematoma involves the interaction between the fibrin and the surface of the dura, and that blood supply from the dura is essential for its development. CSF does not play an important role in clot formation. In 10 dogs, Ohshima also inoculated the subdural space with a blood clot.22 Similar to the results of fresh blood inoculation, CT scans revealed high or mixed density in the initial stages of the clot followed by shrinkage in the chronic stage. In addition, tissue organization progressed from granulation to fibrous connective tissue.
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Subcutaneous clots Watanabe et al. also injected blood–CSF mixtures into subcutaneous pockets created in the abdomen of dogs.23 Fluctuant hematoma masses were produced that tended to increase in size from 10 to 20 days after injection, and then decrease in size in almost all of the 30 dogs. Histology showed a thick, vascular membrane on the subcutaneous side of the hematoma and a thin, less vascular fibrous layer on the fascial side. Simply taking autologous blood, allowing it to clot for 24 hours ex vivo, and then implanting it subcutaneously did not produce an expanding hematoma, suggesting that the CSF contributed to the pathogenesis of the lesions. They removed the fibrin on the surface of blood–CSF clots by treating previously formed blood–CSF clots incubated at 37uC for 24 hours, with plasmin. This prevented formation of an expanding hematoma. Fibrin mixed with thrombin and CSF produced an expanding subcutaneous mass, whereas fibrin plus thrombin alone did not. Thus, fibrin in the presence of CSF seemed to be an important factor in the expansion and encapsulation of the hematoma. The importance of these results to human CSDH is uncertain since current understanding of CSDH does not require any CSF to be present in the subdural space. Apfelbaum et al. also conducted an experiment in 2 dogs to replicate the subcutaneous clot implantation described by Watanabe et al.27 The same methods were used and the abdominal hematomas displayed a well-developed membrane and modest swelling 2 weeks after implantation. In cats, Apfelbaum et al. demonstrated that subcutaneous injections of the clots into pockets in the abdomen slowly resolved using previously described methods.23 Clots were surrounded by well-described membranes, as seen in other experiments, although the expansions seen by Watanabe et al. were absent. CSF did not have any effect on subcutaneous clot absorption and clots never approached the sizes reported by Watanabe et al.23 Labadie and Glover conducted numerous experiments injecting or surgically implanting various blood products and chemicals into the subcutaneous space over the dorsal spine of rats.29 Three-monthold human platelet-free plasma was mixed with thrombin and then, in some cases, CSF and then implanted through a surgical incision or injected into the subcutaneous space. Some of these clots (without CSF) were injected along with carrageenan, the inflammatory extract from the seaweed Chondrus crispus. Autologous rat blood mixed with thrombin, or frozen and thawed and then mixed with thrombin, also was injected in other experiments. The hematomas were aspirated 9 days after implantation, fluid obtained was analyzed for various factors and the
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animals were euthanized and examined histologically. All of the injected hematomas decreased in size for the first 2 days but 43% of rats with implanted clots and 33% of rats injected with platelet-free plasma showed growth of the subcutaneous masses after 5 or 6 days. Adding CSF made no difference. A multilayered fibroblastic membrane with neovascularity surrounded the cavities and was similar to human CSDH. Autologous hemolyzed (frozen and then thawed) blood became liquified and enlarged in 47% of the injected animals. The cavities were filled with brown or dark yellow fluid resembling that seen in human CSDH. Plasma alone did not produce an enlarging mass unless carrageenan was added, in which case 33–50% enlarged. The hematoma fluid contained fibrin degradation products, hemoglobin, albumin, gamma globulin and protein, but the osmolality was the same as serum. It was determined from these results that plasma provides the fibrin matrix that allows the enlargement of the clot. The inflammatory reaction resulting from leukocyte, erythrocyte, hemoglobin, platelet and fibrin breakdown products was also thought to be a stimulus for growth. The production of an enlarging subcutaneous hematoma seemed to depend on injecting large volumes of hemolyzed blood or fresh whole blood clotted in situ. The same investigators injected autologous blood clotted by adding thrombin, into the dorsal subcutaneous space of rats (n533) and treated 16 with daily injections of intramuscular dexamethasone.30 Animals were euthanized after 9 days. Forty-seven percent of the control animals developed expanding hematomas whereas none of the animals treated with dexamethasone did. Treatment with dexamethasone also inhibited neomembrane formation histologically.
Vessel avulsion models In 7 dogs, Goodell and Mealey made parietal parasagittal burr holes and silk sutures were placed around cortical bridging veins and carried through the dura before closing the incision.21 The bridging veins were torn after 3 days by pulling out the sutures, once again, after administration of urea. Of the 4 surviving dogs with torn bridging veins, two euthanized after 11–21 days showed organization and reabsorption of the hemorrhage and two euthanized at days 33 and 72 had no observable abnormalities.
Spontaneous hematoma models Yashon et al. reported an autopsy on a 4-monthold Chihuahua with congenital hydrocephalus and a CSDH.31 Asakawa et al. also described asymmetric brain atrophy with a CSDH that accompanied neuronal ceroid lipofuscinosis in an 11-month-old Dachshund.32
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Mice were given a single intraperitoneal injection of 6-aminonicotinamide (6-AN), a niacin antagonist and neurotoxin, on the fifth postnatal day.33 Mice were sacrificed up to 30 days after the injections. By the 9th day post-injection, 37 of 38 mice exhibited hydrocephalus, neurological abnormalities and developmental deficits. On the 20th to 23rd day postinjection, approximately 60% developed spontaneous subdural hemorrhages in the parietal and occipital regions. The authors observed organizing subdural blood clots in some cases surrounded by brownish blood clots. The occipital cortex was noted to become membrane-thin and perforated at the occipital horns. Histological observations also confirmed similarities to human CSDH with a thin inner membrane of several cell layers and a thick outer membrane with fibroblasts, neovascularity, hemosiderin-laden macrophages and fresh hemorrhage. The authors hypothesized that the hematomas formed when the occipital horns of the lateral ventricles perforated into the subdural space and the ventricles collapsed, tearing bridging veins and causing subdural bleeding.
Discussion Few studies attempting to model CSDH experimentally have been conducted since 1987, perhaps because existing models have been unsuccessful at replicating this pathology. For these reasons we have reviewed the past and present literature on CSDH, as it is important to understand the strengths and weaknesses of current models so that an effective model can be established in the future. Attempts to create an experimental model of human CSDH have been made in mice, rats, cats, dogs and monkeys. Intracranial studies have been done in all of these species except rats. Injecting various blood products and mixtures into the subcutaneous space of rats, cats and dogs has produced transiently expanding, liquified hematomas in up to half of the animals. The histology of the outer CSDH membrane in humans resembles closely the wall of the hematomas produced by subcutaneous injection of blood and is well-described as the typical response of the body to a hematoma.27 Most hematomas resolved spontaneously. It is well known that chronic expanding hematomas develop in parts of the human body, in the subdural space and even in the brain itself.34,35 The histology of the walls of these lesions is very similar to CSDH. Despite the past studies on CSDH, a reproducible model has not yet been reported. There are a number of limitations with the published models. No reproducible model of a true continually expanding, liquified hematoma that does not resolve spontaneously has been described. Modification of the existing ASDH models may be feasible but rapid resolution of the clot has generally
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occurred in such models, which is also a common problem in CSDH models. Subcutaneous blood injections are of unknown relevance to human CSDH. Whether the pathophysiology of the response to blood in the subcutaneous space is the same as in the subdural space is unknown. The main treatment for CSDH is surgical drainage. Since this leads to faster resolution of human CSDH, it might be the gold standard against which the applicability of an animal model is tested. Since none of the models produces a chronically expanding lesion, the endpoint should have to be faster resolution with drainage compared to without. Head injury precedes CSDH in 60–80% of cases and this is widely believed to be the etiologic factor. However, there are inconsistencies. What is the cause in those with no recollection of trauma? Was the trauma so minor as to be forgotten or are there some other factors? If the first event is tearing of a bridging vein that can occur after such minor injury that it is forgotten, then there should be blood visible on brain imaging right after the injury. However, the senior author has imaged a number of patients where the CT scan is normal right after the injury and a CSDH appears after 2–4 weeks. Finally, a CSDH typically is not near where the bridging veins enter the sinuses, but over the convexity where there are no bridging veins. Therefore, a surgical method aimed at damaging bridging veins may not be a reliable option. Watanabe et al. suggested that fibrin and CSF were essential for creating an expanding, liquified hematoma.23 The continuing expansion of blood plus CSF reported by Watanabe and colleagues was not replicated by Ohshima, who had to treat the dogs with mannitol and heparin.22 However, the findings of Ohshima also have not been replicated. Although Apfelbaum et al. found that CSF was not important for CSDH formation in animal models, human studies have suggested that CSF leakage may be involved in CSDH pathogenesis and recurrence.36 No studies in vitro have examined the role of CSF in clot development. Therefore, further studies must be done to discern the role of CSF in the pathogenesis of CSDH. Brain atrophy is believed to be a predisposing factor to CSDH, given that patients with high cortical atrophy have an increased risk of CSDH. Volumetric analysis of CT brain scans has established an association between the two.37 Cerebral atrophy may facilitate dura–arachnoid separation after trauma and has been shown in cats and dogs to cause fibrininduced proliferation of granulation tissue on the dura.27 The methods used by Watanabe et al., in which a balloon was utilized to create a space for blood injection, mimics the dura–arachnoid separation seen in CSDH patients with cerebral atrophy.23 This model may be especially beneficial for studying the effect of
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brain atrophy on CSDH in animals and can lead to a further understanding as to why there is an increased risk for CSDH in older patients with cerebral atrophy. The pathogenesis of CSDH enlargement also has been attributed to various factors including osmotic and oncotic gradients, fibrinolytic activity in the fluid, inflammation, angiogenesis and bleeding from the outer membrane.38 A discussion on the pathogenesis is not the topic of this review, but we suggest that development of an animal model might facilitate understanding of the pathogenesis of human CSDH. The results of this review suggest that this has not been accomplished yet. Studies in the early twentieth century were performed on larger animals, which tended to be used more commonly in experiments compared to now. However, experiments in large animals can be expensive and pose limitations due to insufficient information about and inability to manipulate their genomes. It may be more feasible to first establish a reproducible model in smaller animals, such as rodents, and then attempt CSDH models in larger species. Mouse models are advantageous due to extensive knowledge of their genome, similarity to human physiology, accelerated lifespan, cost effectiveness and ability to manipulate the genome. Rodent models have been widely used to replicating the pathophysiology of other human diseases, such as subarachnoid hemorrhage.39–42 Compared to the subarachnoid space targeted in these studies, the subdural space is relatively easy to access in mice. Thus, rodent models of CSDH may be one avenue to explore initially. A model that replicates the characteristics of human CSDH is essential; this includes a persistent, expanding, liquefied hematoma encased in a neomembrane. Since CSDH is most prevalent in the elderly and males,1,43 future studies may benefit from the use of older male animals. Given that CSDH is a result of an autologous, spontaneous hemorrhage into the potential subdural space, it may be beneficial to use a model that simulates all aspects of CSDH pathogenesis. This may be achieved by utilizing fresh blood injection into the subdural space rather than subdural or subcutaneous clot placement. A model in which bridging veins are weakened followed by a form of mild head trauma may also be a novel method of inducing spontaneous CSDH. Future CSDH research should focus on establishing a reproducible animal model of CSDH as it is essential for improving treatment methods and, ultimately, quality of life for CSDH patients.
Disclosures R. L. Macdonald receives grant support from the Physicians Services Incorporated Foundation,
Experimental Models of Chronic Subdural Hematoma
Brain Aneurysm Foundation, Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada. R. L. Macdonald is a consultant for Actelion Pharmaceuticals and Chief Scientific Officer of Edge Therapeutics, Inc.
Acknowledgements None.
References 1 Tanaka Y, Ohno K. Chronic subdural hematoma-an up-to-date concept. J Med Dent Sci. 2013;60:55–61. 2 Fogelholm R, Waltimo O. Epidemiology of chronic subdural haematoma. Acta Neurochir (Wien). 1975;32:247–50. 3 Kudo H, Kuwamura K, Izawa I, Sawa H, Tamaki N. Chronic subdural hematoma in elderly people: present status on awaji island and epidemiological prospect. Neurol Med Chir (Tokyo). 1992;32:207–9. 4 Chen JC, Levy ML. Causes, epidemiology, and risk factors of chronic subdural hematoma. Neurosurg Clin N Am. 2000;11:399–406. 5 Santarius T, Kirkpatrick PJ, Ganesan D, Chia HL, Jalloh I, Smielewski P, et al. Use of drains versus no drains after burrhole evacuation of chronic subdural haematoma: a randomised controlled trial. Lancet. 2009;374:1067–73. 6 Kinsella k, Velkoff VA. An aging world: 2001. U.S. Government Printing Office, Washington DC. 2001. 7 Svetlichnaya J, Klein L. Atrial fibrillation in elderly patients with heart failure: convergence of two cardiovascular epidemics in the 21st century. Expert Rev Cardiovasc Ther. 2011;9: 903–12. 8 Weigel R, Schmiedek P, Krauss JK. Outcome of contemporary surgery for chronic subdural haematoma: evidence based review. J Neurol Neurosurg Psychiatry. 2003;74:937–43. 9 Miranda LB, Braxton E, Hobbs J, Quigley MR. Chronic subdural hematoma in the elderly: not a benign disease. J Neurosurg. 2011;114:72–6. 10 Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gotzsche PC, Ioannidis JP, et al. The prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. PLoS Med. 2009;6:e1000100. 11 Putnam TJ, Putnam IK. The experimental study of pachymeningitis hemorrhagica. J Nerv Ment Dis. 1927;65:260–2. 12 Serres A. Nouvelle division des apoplexies. Annuaire Med Chir des Hop. 1819;1:246. 13 Laborde J. Contribution a l’etude des conditions pathogeniques des kystes sanguines de l’arachnoide; recherches experimentales sur les animaux. C r Soc de biol Paris. 1864;1:70. 14 Sperling H. Ueber pachymeningitis haemorrhagica interna. Inaug.-Diss., Univ. Konigsberg. 1872. 15 Van Vleuten CF. Ueber pachymeningitis haemorrhagica interna traumatica. Inaug.-Diss., Univ. Bonn. 1898. 16 Virchow R. Das haematom der dura mater. Verchaudlungen der Phys Medizin Gesellschaft zu Wurzburg. 1857;7:134–42. 17 Kremiansky J. Ueber die pachymeningitis interna haemorrhagica bie menschen u. Hunden Arch f Path Anat. 1868;42: 29–161. 18 Saltykow. Referat, alcoholismus chronicus. Centralbl f allg path u path anat. 1911;22:849. 19 Gardner WJ. Traumatic subdural hematoma with particular reference to the latent interval. Arch. Neurol. Psychiatry. 1932;27:847–58. 20 Christensen E. Studies on chronic subdural hematoma. Acta Psychiatr Neurol. 1944;19:69–148. 21 Goodell CL, Mealey J Jr. Pathogenesis of chronic subdural hematoma. Experimental studies. Arch Neurol. 1963;8:429–37. 22 Ohshima T. Experimental study on the evolution of chronic subdural hematoma. Neurol Med Chir (Tokyo). 1982;22:696– 706. 23 Watanabe S, Shimada H, Ishii S. Production of clinical form of chronic subdural hematoma in experimental animals. J Neurosurg. 1972;37:552–61. 24 Eijkenboom M, Gerlach I, Barker A, Luiten P, van der Staay FJ. Chronic cognitive effects of bilateral subdural haematomas in the rat. Neuroscience. 2004;124:523–33.
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25 Miller JD, Bullock R, Graham DI, Chen MH, Teasdale GM. Ischemic brain damage in a model of acute subdural hematoma. Neurosurgery. 1990;27:433–9. 26 Jussen D, Papaioannou C, Heimann A, Kempski O, Alessandri B. Effects of hypertonic/hyperoncotic treatment and surgical evacuation after acute subdural hematoma in rats. Crit Care Med. 2008;36:543–9. 27 Apfelbaum RI, Guthkelch AN, Shulman K. Experimental production of subdural hematomas. J Neurosurg. 1974;40:336–46. 28 Weir B. The osmolality of subdural hematoma fluid. J Neurosurg. 1971;34:528–33. 29 Labadie EL, Glover D. Physiopathogenesis of subdural hematomas. Part 1: histological and biochemical comparisons of subcutaneous hematoma in rats with subdural hematoma in man. J Neurosurg. 1976;45:382–92. 30 Glover D, Labadie EL. Physiopathogenesis of subdural hematomas. Part 2: inhibition of growth of experimental hematomas with dexamethasone. J Neurosurg. 1976;45:393–7. 31 Yashon D, Small E, Jane JA. Congenital hydrocephalus and chronic subdural hematoma in a dog. J Am Vet Med Assoc. 1965;147:832–6. 32 Asakawa MG, MacKillop E, Olby NJ, Robertson ID, Cullen JM. Imaging diagnosis–neuronal ceroid lipofucinosis with a chronic subdural hematoma. Vet Radiol Ultrasound. 2010;51:155–8. 33 Aikawa H, Suzuki K. Experimental chronic subdural hematoma in mice. Gross morphology and light microscopic observations. J Neurosurg. 1987;67:710–6. 34 Kunisawa S, Kosaka S, Matsukura T, Nakashima T, Okabayashi T, Miyagawa H, et al. Chronic expanding hematoma in sternum resected 5 years after median sternotomy. Ann Thorac Surg. 2008;85:1447–8.
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35 Reid JD, Kommareddi S, Lankerani M, Moonja CP. Chronic expanding hematomas. A clinicopathological entity. J Am Med Assoc. 1980;244:2441–2. 36 Kristof RA, Grimm JM, Stoffel-Wagner B. Cerebrospinal fluid leakage into the subdural space: possible influence on the pathogenesis and recurrence frequency of chronic subdural hematoma and subdural hygroma. J Neurosurg. 2008;108: 275–80. 37 Yang AI, Balser DS, Mikheev A, Offen S, Huang JH, Babb J, et al. Cerebral atrophy is associated with development of chronic subdural haematoma. Brain Inj. 2012;26:1731–6. 38 Stoodley M, Weir B. Contents of chronic subdural hematoma. Neurosurg Clin N Am. 2000;11:425–34. 39 Sabri M, Ai J, Lass E, D’Abbondanza J, Macdonald RL. Genetic elimination of enos reduces secondary complications of experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2013;33:1008–14. 40 Sabri M, Jeon H, Ai J, Tariq A, Shang X, Chen G, et al. Anterior circulation mouse model of subarachnoid hemorrhage. Brain Res. 2009;1295:179–85. 41 Friedrich V, Flores R, Muller A, Bi W, Peerschke EI, Sehba FA. Reduction of neutrophil activity decreases early microvascular injury after subarachnoid haemorrhage. J Neuroinflammation. 2011;8:103. 42 Sehba FA, Friedrich V. Cerebral microvasculature is an early target of subarachnoid hemorrhage. Acta Neurochir Suppl. 2013;115:199–205. 43 Kanat A, Kayaci S, Yazar U, Kazdal H, Terzi Y. Chronic subdural hematoma in adults: why does it occur more often in males than females? Influence of patient’s sexual gender on occurrence. J Neurosurg Sci. 2010;54:99–103.
Experimental models of chronic subdural hematoma Josephine A. D’Abbondanza1,2, R. Loch Macdonald1,2 1
Division of Neurosurgery, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario, Canada, 2 Department of Surgery, Institute of Medical Science, University of Toronto, Ontario, Canada Objectives: Chronic subdural hematoma (CSDH) is a common neurosurgical problem. Most studies of pathogenesis and treatment involve humans. Advances in understanding of human diseases may be made using animal models. We reviewed all animal models of CSDH and report here their results, conclusions and limitations in order to set a baseline upon which further advanced experimental work related to this disease can be made. Methods: PubMed, Medline, Embase and ISI Web of Knowledge were searched with no time limits using the keyword ‘chronic subdural hematoma’ and MeSH term ‘hematoma, subdural, chronic’. The authors reviewed all papers written related to this disease and selected all publications involving animals. There were no other restrictions. The findings and conclusions of the papers are summarized here. No formal analysis was done because of the variation in species used, methods for induction of CSDH, times of assessment and reporting of results. Results: Attempts to create CSDH have been made in mice, rats, cats, dogs and monkeys. Methods include injection or surgical implantation of clotted blood or various other blood products and mixtures into the potential subdural space or the subcutaneous space. No intracranial model produced a progressively expanding CSDH. Transient hematoma expansion with liquification could be produced by subcutaneous injections in some models. Spontaneous subdural blood collections were found after creation of hydrocephalus in mice by systemic injection of the neurotoxin, 6-aminonicotinamide. The histology of the hematoma membranes in several models resembles the appearance in humans. None of the models has been replicated since its first description. Discussion: We did not find a report of a reproducible, well-described animal model of human CSDH. Keywords: Chronic subdural hematoma, Experimental model
Introduction Chronic subdural hematoma (CSDH) is a form of hematoma occurring between the dura and arachnoid mater. It is hypothesized to be caused by traumatic brain injury, where shearing forces cause bridging veins to tear where they traverse the potential subdural space and enter the dura.1 Chronic subdural hematoma is more common with an increase in age. Other factors associated with development of CSDH are chronic alcohol abuse, ventriculoperitoneal shunting and other factors associated with cerebral atrophy. Chronic subdural hematoma generally develops a few days to weeks after minor trauma, but these incidents are not documented in up to 50% of the patients. The clinical presentation can be acute,
Correspondence to: Josephine A. D’Abbondanza, Division of Neurosurgery, Labatt Family Centre of Excellence in Brain Injury and Trauma Research, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 209 Victoria Street, Toronto, Ontario M5B 1T8, Canada. Email: [email protected]
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however, it is usually secondary to acute bleeding into the CSDH. Chronic subdural hematoma differs from acute subdural hematoma (ASDH) in terms of the speed of onset, as ASDH develops much faster after head injury.1 Bleeding in both ASDH and CSDH is usually venous and subsequently slower than arterial epidural hemorrhage.1 Chronic subdural hematoma is common in neurosurgical practice and it is understudied relative to its prevalence. Two studies estimated the incidence. Fogelholm and Waltimo reported that the incidence of CSDH in Helsinki between 1967 and 1973 was 1.7 per 100 000 per year, with a steep rise in incidence with an increase in age.2 Kudo et al. found an overall incidence of 13 per 100 000 per year in Awaji Island, Japan, based on the data collected between 1986 and 1988.3 Again there was a marked increase with an increase in age. The mean age of those affected with CSDH is not well documented other than in multiple, mostly retrospective, case series where the
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mean age ranged from 50 to 67 in a review of eight case series4 and about 75-years-old in a study from the United Kingdom.5 Males constitute about 75% of cases.5 Given that the proportion of people over 65-years-old is predicted to double worldwide between 2000 and 2030, CSDH will become an even more frequent health care problem.6 The expanding indications for anticoagulation in the elderly also may increase the incidence.7 While the treatment is relatively simple, the disease is associated with substantial morbidity, including recurrence rates of 5–30%.8 Mortality 6 months after treatment was 13% (28 of 210 patients) in a clinical trial that accrued patients between 2004 and 20075 and 26% (55 of 209 patients) in a series of cases collected since 2000.9 Although many studies have focused on clinical treatment and a few on pathogenesis and pathology in humans, there has been less attention to experimental models and investigation of CSDH. While the study of humans is of great interest, an animal model could lead to pathophysiologic insights and perhaps new treatments. The purpose of this paper is to review animal models of CSDH. We did not find guidelines for systematic reviews of animal models of diseases but we began by following the PRISMA structure.10 The rationale for this review is to summarize what has been previously attempted for the experimental study of CSDH, so that those interested in developing CSDH models or in studying CSDH have a baseline upon which they can move forward. The objective of this article was to summarize every study attempting to or actually creating an experimental model of CSDH.
Methods PubMed, Medline, Embase and ISI Web of Knowledge were searched in September 2013 with no date limits. The keyword was ‘chronic subdural hematoma’. The MeSH term ‘hematoma, subdural, chronic’ was also included. The titles, abstracts and key words of every paper related to this disease were searched and then papers were selected from this search and from the reference lists of the reviewed papers. There was no review protocol. The only eligibility criterion was that animals were included in the paper. There was no language restriction. The two authors performed the above process individually and then collected the data on the species used, methods, results and conclusions of the included papers. Both authors summarized the data: the first author wrote the narrative description and the second author revised it. No formal analysis was done because of the variation in species used, methods for induction of CSDH, times of assessment,
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lack of quantitative data and variable reporting of results.
Results The initial search showed 2997 references. The titles and abstracts were reviewed and five publications were found that used animals to study CSDH. None of the remaining reports was an animal study. The reference lists of the five papers were searched, which disclosed an additional eight papers. Finally, a selection of the 2997 references (241) were read and the reference lists reviewed to see if there are any other studies involving animals. This found no additional papers. There are a number of papers found that were published more than 85 years ago that did not provide any useful information; in these papers the species, surgical procedure or results were not mentioned or lacked sufficient details for analysis. They are summarized for historical interest because they show that some current understanding, whether correct or not, originated from those papers. The more recent included studies are in the tables (Tables 1–4).
Early models Putnam and Putnam reviewed studies attempting to produce CSDH in animals.11 Serres tore the longitudinal sinus subdurally in animals and noted that the animals developed seizures and hemiplegia within hours.12 When the animals were euthanized within a few days, there was liquid blood in a fibrinous sac that was adherent to the dura. These observations within days of hemorrhage would not represent CSDH. Similarly, Laborde injected subdural blood in dogs and cats but sacrificed them after a day.13 Sperling and others created trephines in the skulls of rabbits and injected whole blood under the dura.14,15 When animals were sacrificed after 4 days to 6 weeks, a thin, brown, vascular membrane was almost always found on the inner surface of the dura, likely representing organization of the hematoma. In humans, CSDH sometimes has a loculated appearance. Inflammation of the dura, theoretically initiated by blood and fibrin exposure, results in the formation of a neomembrane around a liquified clot.1 Other processes postulated to contribute to the pathogenesis of CSDH are fibrinolytic activity causing repeated hemorrhage into the CSDH. Sperling does not describe a liquified hematoma and he provided no illustrations of the membrane; therefore, it is difficult to infer similarities to human CSDH solely by his descriptions. He concluded that CSDH was produced by capillary or venous hemorrhage into the subdural space. This was in contrast to the prevailing theory at that time, which states that CSDH was an inflammatory lesion, possibly due to infection.16
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1963
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Christensen
Goodell and Mealey
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1927
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Gardner
Year
Author
Dog (n 5 9)
Dog (n 5 2–4 per group)
Dog (n 5 7)
Dog (n 5 5)
Dog (n 5 3), cat (n 5 15)
Species
Subdural injection, with or without urea to reduce brain volume and allow injection of more blood
Subdural injection with or without ligation of superior sagittal sinus, ligation of superior sagittal sinus alone
Subdural injection to the contralateral subdural space with a curved needle through a 1–2 cm trephine
Subdural injection through a 1–2 cm trephine
Subdural injection through a 1–2 cm trephine
Method of injection
3.8% citrate blood or paraffin (to prevent clotting) blood, also tried repeated blood injections and blood injection plus head injury, volume not stated
Autologous fresh blood
Subdural space
Autologous unclotted whole blood
Subdural space
Subdural space
Autologous unclotted whole blood
Autologous whole blood, defibrinated blood, washed fibrin
Substances injected/volume
Subdural space
Subdural space
Site of injection
Table 1 Blood injection models of chronic subdural hematoma (CSDH)
2–12 ml once, repeated injections weekly of up to 27 ml
Not stated
3– 11.5 ml
0.7–3 ml
1–2 ml
Volumes
Up to 84 days
8 days to 5 weeks
Not stated
3–14 weeks
1 day to 3 months
Duration of study
Subdural hematomas that organized and resolved over time
No gross evidence of injected blood if euthanasia was more than couple of weeks after injection Residual organizing hematoma with membranes that resolved with time
No gross evidence of injected blood
Small remaining volumes of subdural blood, occasional membranes
Gross pathology
No evidence of subdural hematoma in most animals, some showed a fibrous subdural membrane
Outer membrane of fibroblasts, capillaries, large vascular channels, organizing hematoma with no liquidcontaining cavity
Not mentioned
Not mentioned
Outer membrane of granulation tissue with neovascularity, organizing hematoma, thin inner mesothelial membrane
Microscopy
Chronic subdural hematoma is of traumatic origin, ligating the superior sagittal sinus slowed clearance of subdural blood, this model was similar to human CSDH except that the small volumes of blood injected completely organized rather than liquifying Subdural blood alone was insufficient to produce a CSDH, the osmotic theory is untenable
Pachymeningitis could be produced by injection of blood or fibrin or by chronic alcohol intake, but progressive lesions like in humans could not be created It is difficult if not impossible to reproduce a CSDH in dogs, ascribed to the osmotic theory of hematoma enlargement It is difficult if not impossible to reproduce a CSDH in dogs, ascribed to the osmotic theory of hematoma enlargement
Conclusions
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2004
Eijkenboom
2008
1982
Ohshima
Jussen
Year
Continued
Author
Table 1
Rat (n 5 56)
Rat (n 5 115 in five groups)
Dog (n 5 45)
Species
Subdural space
Subdural space
Subdural injection after craniotomy
Subdural injection after craniotomy
Subdural space
Site of injection
Subdural injection after inflating a balloon in the subdural space to create a cavity
Method of injection
Autologous fresh blood
Autologous fresh blood
Autologous clotted blood, fresh blood, blood mixed with cerebrospinal fluid, blood mixed with cerebrospinal fluid plus systemic mannitol and heparin
Substances injected/volume
4 ml
1 ml bilaterally
3–4 ml
Volumes
Up to 12 days
1–18 weeks
Up to 21 days
Duration of study
Clot removed after 60 minutes and local cerebral blood flow was similar across all groups
Computed tomography (CT) showed resolving hematomas if only fresh or clotted blood was injected, mixing with cerebrospinal fluid (CSF) lowers the density of the hematoma on CT but it still resolved, 3 of 10 dogs treated with blood plus heparin plus mannitol showed expanding hematomas until euthanasia in 21 days Hematomas observed in the sensorimotor cortex and other lesions, including ventricle dilations and compression damage
Gross pathology
Calcium deposits in thalamic nuclei, astrocyte reactivity evident early followed by the disappearance at week 8 and strong reappearance at week 18 Contralateral infarction and neurological scores due to hematoma were reduced with treatment
The outer membrane of granulation tissue, capillaries, expansion was due to hemorrhage from the outer membrane neovascularity
Microscopy
Hypertonic/hyperoncotic treatment (HHT) and surgical evacuation reduced intracranial pressure improved functional outcome
Bilateral subdural hematoma caused impairment in spatial navigation that recovered by week 8 but reoccurred at week 18
A combination of blood plus cerebrospinal fluid plus recurrent hemorrhage from the outer membrane needed to form a chronic expanding hematoma
Conclusions
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1974 Dog Subdural or Subdural or (n 5 2) and subcutaneous subcutaneous cat (n 5 40) injection, space polyethylene sheet between dura and hematoma and devascularization of dura by excision and reinsertion
Apfelbaum et al.
Subdural or subcutaneous space
Subdural space
Site of injection
1972 Dog (n 5 62), monkey (n 5 5)
Subdural injection, with or without urea to reduce the brain volume and allow injection of more blood, some animals had cisternopleural shunts Subdural injection after inflating a balloon in the subdural space to create a cavity, or subcutaneous injection
Method of injection
Watanabe et al.
Goodell 1963 Dog and Mealey (n 5 52 in six groups)
Author
NO .
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Autologous blood with or without cerebrospinal fluid, defibrinated blood
Autologous clotted blood with or without cerebrospinal fluid
Autologous clot, with or without cerebrospinal fluid, streptokinase, or hydrocortisone, frozen and thawed blood
Substances injected/volume
Table 2 Clot injection and placement models of chronic subdural hematoma (CSDH) Duration of study
1–5 ml
8–30 days
7–14 days in dogs, 14–21 days in monkeys
Up to 2–12 ml once, 84 days repeated injections weekly of up to 27 ml
Volumes
Subdural hematomas that organized and resolved over time
Three dogs developed enlarging subdural fluid collections, the rest showed resolving hematomas
Subdural hematomas that organized and resolved over time
Gross pathology
The outer membrane with sinusoidal vascular channels lined by a single layer of endothelial cells, loose connective tissue, macrophages, fibroblasts and inflammatory cells, thin inner membrane. Similar histology in the subcutaneous space with some dogs developing expanding hematomas, removing fibrin from the clots with plasmin or implanting fibrin plus thrombin did not cause an expanding hematoma, only expanding hematomas were with blood containing fibrin plus cerebrospinal fluid Outer membranes with fibroblasts, neovascularity, extravasation of erythrocytes was observed from the neovascularity, thin inner membrane, liquifying hematoma
The outer membrane with neovascularity, fibroblasts, phagocytes, organizing hematoma and thin, single cell layer inner membrane, injection of other substances with the blood did not affect the findings
Microscopy
Cerebrospinal fluid is not necessary for CSDH formation but fibrin is, the histologic response to extravascular blood clot is the same in the subdural space as in other body spaces, CSDH expansion is due to bleeding from the neovascularity in the membranes
A combination of blood containing fibrin plus cerebrospinal fluid is necessary to produce an expanding fluid collection
Subdural blood alone was insufficient to produce a CSDH, the osmotic theory is untenable
Conclusions
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1976 Rat (n 5 154)
Labadie and Glover
Subcutaneous implantation or injection
Method of injection
Substances injected/volume
Subcutaneous Human plasma, space plasma plus cerebrospinal fluid, plasma plus carrageenan, autologous hemolyzed blood, autologous fresh blood
Site of injection
Year
1963
Author
Goodell and Mealey
Dog (n 5 7)
Species
Avulsion of bridging veins
Method of injection No blood injected
Site of injection
Volumes 9 days
No blood injected
Microscopy
Up to 72 days
Subdural hematomas that organized and resolved over time
Gross pathology
Surviving animals showed reabsorption or no evidence of hematoma/fibrous membrane
Microscopy
Most hematomas Fibroblastic resolved, hematomas membrane with that expanded with neovascularity liquified contents between about 10 and 20 days were larger volumes of plasma alone if surgically implanted (43%) but not if injected (0%), plasma plus carrageenan (33–50%), autologous hemolyzed or whole blood 45–50%)
Gross pathology
Duration of study
Duration of study
Volumes
2–16 ml
Tearing of bridging veins using silk sutures, intravenous hypertonic urea or heparin
Substances injected/volume
Table 3 Vessel avulsion models of chronic subdural hematoma (CSDH)
Year
Author
Species
Continued
Table 2
Subdural blood from torn bridging veins was insufficient to produce a CSDH, the bridging vein theory is untenable
Conclusions
The composition and volume of clot are important for developing an expanding hematoma, plasma–fibrin provides the matrix shape for the hematoma, membrane formation requires erythrocytes, hemoglobin, leucocytes, other blood elements and inflammation whereas cerebrospinal fluid is not necessary
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Conclusions
Hematomas formed when the ventricular system spontaneously decompressed into the subdural space, collapsing the cortical mantle and tearing bridging veins Hematomas surrounded by a thin inner membrane and a thick outer membrane with fibroblasts, neovascularity, hemosiderin-laden macrophages and fresh hemorrhage No blood injected
Creation of hydrocephalus in neonatal mice by intraperitoneal injection of 6aminonicotinamide
No blood injected
Up to 30 days
Hydrocephalus, 60% developed spontaneous subdural hematomas
Putnam and Putnam created 1–2 cm trephines at the junction of the longitudinal and transverse sinuses in dogs and cats and injected autologous blood into the subdural space.11 Sometimes the blood was inadvertently injected into the subarachnoid space. The blood also had a tendency to leak out through the needle hole. In an attempt to correct this, washed fibrin was injected into some animals. The animals were sacrificed after 1–50 days. In some cases it was possible to produce a thin neomembrane adherent to the dura that overlay a hematoma. However, the border between the membrane and clot was not as distinct as in human CSDH and there was no liquified hematoma cavity. Empty mesothelial-lined spaces, similar to those in human outer CSDH membranes, were found throughout the membrane and were sometimes found lining the dura. Gardner subjected five dogs to subdural injection of autologous unclotted blood and euthanasia after 3–14 weeks.19 Little or no blood was visible beneath the dura of these animals. He also made a burr hole on one side of the head in seven dogs and inserted a curved needle through the hole and across the midline to the contralateral convexity subdural space and injected 3–11.5 ml unclotted autologous blood. When animals were euthanized more than a couple of weeks after there were no hematomas remaining. Additionally, Gardner placed cellophane sacs containing autologous whole blood into the subdural space of eight dogs. The sacs were removed after 3–18 days and were found to be 39–103% heavier. This was taken as evidence that an osmotic gradient (high osmolarity in the hematoma compared to the cerebrospinal fluid [CSF]) caused the hematoma to imbibe fluid and create a CSDH. Christensen performed subdural injections of citrated blood in dogs by sacrificing them after 8–11 days.20 Blood was injected every week for 3 weeks in two dogs and the animals were euthanized
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Other investigators injected a mixture of fatty acids into the subdural space and produced dural thickening and occasionally a laminated hemorrhage.11 None of these studies was accompanied by histology. Physicians in the nineteenth century recognized that CDSH or pachymeningitis hemorrhagica interna, as it was called, was more common in alcoholics. Kremiansky conducted experiments on dogs, feeding them brandy every day for 2 or 3 months. He found a pachymeningitis in 3 out of 4 experiments and concluded that alcohol caused inflammation that caused the subdural lesions.17 His experiments on dogs have been repeated by a number of investigators, and subdural membranes have occasionally, although usually not, been produced in animals.18
Blood injection models
Mouse (n 5 38)
Substances injected/volume Site of injection Year
Species
Method of injection
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Table 4 Spontaneous hematoma models of chronic subdural hematoma (CSDH)
Volumes
Duration of study
Gross pathology
Microscopy
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1 week after the last injection. How the injections were performed is not stated. There was proliferation of blood vessels and fibrous tissue forming a structure similar to the outer membrane of a CSDH. The thick outer membrane produced consisted of fibroblasts, capillaries up to 50 mm in diameter (similar to the large sinusoidal vessels seen in human outer CSDH membranes) and other cells. Deep to this was some residual blood with decaying erythrocytes and infiltrating macrophages but no liquified hematoma cavity. In this investigator experiments, injecting subdural blood and then inflicting head trauma weekly for 3 weeks also did not produce a CSDH. Finally, in four dogs, the superior sagittal sinus was ligated and subdural paraffin (to reduce clotting) blood injected bilaterally into the subdural space. After 11 days to 3 weeks, during euthanasia, histology showed some larger cavities in the subdural space, in addition to the typical outer membrane with neovascularization and fibroblasts. He concluded that injection of subdural blood along with ligation of the superior sagittal sinus in dogs produced a lesion similar to CSDH. In 1963, Goodell and Mealey produced CSDH by injecting 2–10 ml autologous blood subdurally through a burr hole.21 A needle and syringe or a polyethylene catheter was sealed in the subdural space so that blood injected did not leak out of the dural opening into the epidural space. This was a problem with previous needle injection methods. Intravenous urea was usually given in order to reduce the intracranial pressure and permit injection of larger blood volumes. Of the eight dogs with subdural blood injections sacrificed after 8–72 days, 1 died acutely, 5 showed no evidence of hematoma formation and 2 had thin residual membrane or dural thickening. Multiple subdural injections (2–5) of whole blood were administered over 12–35 days to eight dogs. Five survived and exhibited the same pattern of organization and resolution of the blood as seen after single injections, without formation of a CSDH. The investigators recognized the role of craniocerebral disproportion in producing a CSDH and attempted to induce this by injecting subdural blood in dogs with cisternopleural shunts or by administering urea to the dogs for days after the injections. No change in the histology of the hematoma resolution occurred. Subdural injection of blood mixed with CSF, streptokinase, cortisone or frozen and then thawed blood also failed to produce a CSDH. They concluded that subdural blood alone was insufficient to produce a CSDH. They considered the osmotic mechanism of CSDH expansion to be untenable. Ohshima injected fresh autologous blood and blood mixed with CSF into the subdural space of
Experimental Models of Chronic Subdural Hematoma
dogs.22 The blood injections followed the method of Watanabe et al., where a balloon was used to expand the subdural space before the injection.23 In the group injected with blood mixed with CSF, some animals were treated with daily doses of mannitol and heparin. They were imaged with computed tomography (CT) and then examined histologically at various times. All of the hematomas slowly resolved except for 3 of 10 in dogs treated with blood plus CSF, mannitol and heparin, in which the hematomas enlarged over time. Histological examination showed typical outer neovascular, fibrous, thickened membranes that were more marked in dogs injected with blood mixed with CSF. The neomembranes were structurally similar to those seen in human CSDH but were not as expansive. The few animals treated with mannitol and heparin that had expanding hematomas showed evidence of hemorrhage from the neovascular outer membrane. The continuing expansion of blood plus CSF reported by Watanabe et al. was not replicated by Ohshima. Thus, the author concluded that CSF mixing with the blood and hemorrhage from the outer membrane were important in the pathogenesis of CSDH. Eijkenboom et al. induced bilateral subdural hematomas above the somatosensory cortex in rats24 by modifying an ASDH model established by Miller et al. in 1990.25 Briefly, a craniotomy was performed and autologous, non-heparinized venous blood was injected into the subdural space. Hematoma placement was verified 1–2 days after surgery by neurological examination of the animals, and the Morris water maze task was subsequently used to test spatial navigation. Histologically, ventricular dilation and damage to the brain secondary to compression were observed. Impairment in spatial navigation was seen but recovered completely by 8 weeks after surgery. However, 18 weeks after surgery further deterioration was evident and resulted in impaired water maze performance. These contrasting findings at week 8 and week 18 may be an interesting target for future studies. In another study of ASDH by Jussen et al., the chronic effects of hypertonic/hyperoncotic treatment (HHT) and surgical evacuation were also assessed, which may be relevant to studies of CSDH.26 The previously described model by Miller et al. was also used, but for the chronic study, fluids were infused after 30 minutes and subdural blood was evacuated 60 minutes after ASDH. Neurological deficits were assessed on 1–11 days after ASDH and histology was performed on day 12. Arterial blood gases remained constant for the duration of the study. Functional outcome improved over time, but on day 11, the neurological state improved and the infarction size was reduced with HHT alone or when it was
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combined with clot evacuation; there was no additional benefit to the combination treatment.
Clot injection and placement models Subdural clots In 21 dogs, Goodell and Mealey injected clotted autologous blood, from which the serum was removed, into the subdural space via a burr hole.21 Clots were introduced 2–5 times over 12–35 days. Urea and lumbar CSF drainage were used to allow injection of up to 12 ml clot. Pathologic examination over time showed formation of an outer membrane composed of fibroblasts and cells phagocytosing the erythrocytes, a layer of decaying erythrocytes, and an inner membrane that is one cell layer thick, thus resembling the membranes of a CSDH. By 4 weeks there were outer and inner membranes surrounding a collection of proteinaceous fluid and decaying erythrocytes. After 4 weeks, the hematomas resolved. Watanabe et al. tested the effects of subdural injections of autologous blood mixed with CSF incubated ex vivo for 24 hours before implantation.23 Subdural inoculation was performed in 12 dogs by inserting a polyethylene tube connected to a balloon into the subdural space through a 3 cm trephine opening. The balloon was filled slowly, evacuated and then blood plus CSF were injected into the preformed cavity. Three animals developed progressive neurological deficits and were euthanized 7–14 days after the injections. They had loculated, liquified hematomas ranging from 6–10 ml in volume. This was more than the 1–5 ml that was injected initially, suggesting that some hematoma expansion had occurred. In nine dogs, there were no neurological symptoms and the volumes found at euthanasia after 7–14 days were similar to what was injected. The outer membranes in all cases were similar to those seen in humans with numerous sinusoidal, dilated vascular channels lined by a single layer of endothelial cells, loose connective tissue, fibroblasts, macrophages and inflammatory cells. A thin inner membrane was also seen. Watanabe et al. also injected 3–4 ml autologous blood mixed with CSF into the subdural space of 5 monkeys using the same method they used in dogs.23 Three monkeys were sacrificed 14 days and 2 monkeys were sacrificed 21 days after injection. No animals developed neurological symptoms or signs, but one animal euthanized after 21 days had a liquified hematoma much larger than the original injected volume. All hematomas were well encapsulated and histologically similar to those found in the dogs and in humans. Apfelbaum et al. noted the work of Watanabe et al. that suggested that CSF was important in the pathogenesis of CSDH based on the theory that it was drawn osmotically into the hematoma across the inner
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membrane.27 They noted that measurements of the osmolarity of the hematoma fluid in humans showed that the osmotic theory was incorrect.28 In an attempt to replicate the findings of Watanabe et al., Apfelbaum et al. conducted a similar experiment in 2 dogs.27 Clotted blood with or without CSF was implanted by the same method into the subdural space. They were sacrificed on the 13th day following clot injections. The subdural hematomas showed neovascular outer and thin inner membranes with some evidence of fresh bleeding into the hematoma from the outer membrane. CSF had no effect on subdural clot absorption and clots did not reach previously reported sizes.23 Additionally, Apfelbaum et al. created a subdural cavity in cats using the method of Watanabe et al.23 Clots consisting of autologous blood or blood mixed with autologous CSF, artificial CSF or saline were incubated at 37uC for 24 hours ex vivo. They were implanted in the subdural space and animals euthanized at varying times up to 30 days. To identify the factors involved in membrane formation, 10 cats were given additional injections of human or bovine fibrinogen. Four cats also had clots isolated from the dura by a sheet of polyethylene and in another group, a disc of dura was cut out to devascularize it, and then resutured in place and the subdural pocket was created below it. All of the injections produced the same pattern of histologic changes with formation of a thick vascular outer membrane and a thin inner membrane. The outer membrane did not form if the clot was isolated from the dura by polyethylene and was delayed in appearance and thinner if the dura had been devascularized. The authors observed extravasation into the decaying hematoma of intact fresh erythrocytes from the thin-walled sinusoidal channels in the outer membrane. The presence of CSF had no effect. All clots disappeared after 21 days and left behind thickened dura. These findings suggest that CSF is not necessary for membrane formation or clot development. Fibrin seemed essential for the formation of the outer membrane because the outer membrane formed if fibrinogen was implanted subdurally but not if defibrinated blood was. The authors concluded that encapsulation of the hematoma involves the interaction between the fibrin and the surface of the dura, and that blood supply from the dura is essential for its development. CSF does not play an important role in clot formation. In 10 dogs, Ohshima also inoculated the subdural space with a blood clot.22 Similar to the results of fresh blood inoculation, CT scans revealed high or mixed density in the initial stages of the clot followed by shrinkage in the chronic stage. In addition, tissue organization progressed from granulation to fibrous connective tissue.
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Subcutaneous clots Watanabe et al. also injected blood–CSF mixtures into subcutaneous pockets created in the abdomen of dogs.23 Fluctuant hematoma masses were produced that tended to increase in size from 10 to 20 days after injection, and then decrease in size in almost all of the 30 dogs. Histology showed a thick, vascular membrane on the subcutaneous side of the hematoma and a thin, less vascular fibrous layer on the fascial side. Simply taking autologous blood, allowing it to clot for 24 hours ex vivo, and then implanting it subcutaneously did not produce an expanding hematoma, suggesting that the CSF contributed to the pathogenesis of the lesions. They removed the fibrin on the surface of blood–CSF clots by treating previously formed blood–CSF clots incubated at 37uC for 24 hours, with plasmin. This prevented formation of an expanding hematoma. Fibrin mixed with thrombin and CSF produced an expanding subcutaneous mass, whereas fibrin plus thrombin alone did not. Thus, fibrin in the presence of CSF seemed to be an important factor in the expansion and encapsulation of the hematoma. The importance of these results to human CSDH is uncertain since current understanding of CSDH does not require any CSF to be present in the subdural space. Apfelbaum et al. also conducted an experiment in 2 dogs to replicate the subcutaneous clot implantation described by Watanabe et al.27 The same methods were used and the abdominal hematomas displayed a well-developed membrane and modest swelling 2 weeks after implantation. In cats, Apfelbaum et al. demonstrated that subcutaneous injections of the clots into pockets in the abdomen slowly resolved using previously described methods.23 Clots were surrounded by well-described membranes, as seen in other experiments, although the expansions seen by Watanabe et al. were absent. CSF did not have any effect on subcutaneous clot absorption and clots never approached the sizes reported by Watanabe et al.23 Labadie and Glover conducted numerous experiments injecting or surgically implanting various blood products and chemicals into the subcutaneous space over the dorsal spine of rats.29 Three-monthold human platelet-free plasma was mixed with thrombin and then, in some cases, CSF and then implanted through a surgical incision or injected into the subcutaneous space. Some of these clots (without CSF) were injected along with carrageenan, the inflammatory extract from the seaweed Chondrus crispus. Autologous rat blood mixed with thrombin, or frozen and thawed and then mixed with thrombin, also was injected in other experiments. The hematomas were aspirated 9 days after implantation, fluid obtained was analyzed for various factors and the
Experimental Models of Chronic Subdural Hematoma
animals were euthanized and examined histologically. All of the injected hematomas decreased in size for the first 2 days but 43% of rats with implanted clots and 33% of rats injected with platelet-free plasma showed growth of the subcutaneous masses after 5 or 6 days. Adding CSF made no difference. A multilayered fibroblastic membrane with neovascularity surrounded the cavities and was similar to human CSDH. Autologous hemolyzed (frozen and then thawed) blood became liquified and enlarged in 47% of the injected animals. The cavities were filled with brown or dark yellow fluid resembling that seen in human CSDH. Plasma alone did not produce an enlarging mass unless carrageenan was added, in which case 33–50% enlarged. The hematoma fluid contained fibrin degradation products, hemoglobin, albumin, gamma globulin and protein, but the osmolality was the same as serum. It was determined from these results that plasma provides the fibrin matrix that allows the enlargement of the clot. The inflammatory reaction resulting from leukocyte, erythrocyte, hemoglobin, platelet and fibrin breakdown products was also thought to be a stimulus for growth. The production of an enlarging subcutaneous hematoma seemed to depend on injecting large volumes of hemolyzed blood or fresh whole blood clotted in situ. The same investigators injected autologous blood clotted by adding thrombin, into the dorsal subcutaneous space of rats (n533) and treated 16 with daily injections of intramuscular dexamethasone.30 Animals were euthanized after 9 days. Forty-seven percent of the control animals developed expanding hematomas whereas none of the animals treated with dexamethasone did. Treatment with dexamethasone also inhibited neomembrane formation histologically.
Vessel avulsion models In 7 dogs, Goodell and Mealey made parietal parasagittal burr holes and silk sutures were placed around cortical bridging veins and carried through the dura before closing the incision.21 The bridging veins were torn after 3 days by pulling out the sutures, once again, after administration of urea. Of the 4 surviving dogs with torn bridging veins, two euthanized after 11–21 days showed organization and reabsorption of the hemorrhage and two euthanized at days 33 and 72 had no observable abnormalities.
Spontaneous hematoma models Yashon et al. reported an autopsy on a 4-monthold Chihuahua with congenital hydrocephalus and a CSDH.31 Asakawa et al. also described asymmetric brain atrophy with a CSDH that accompanied neuronal ceroid lipofuscinosis in an 11-month-old Dachshund.32
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Mice were given a single intraperitoneal injection of 6-aminonicotinamide (6-AN), a niacin antagonist and neurotoxin, on the fifth postnatal day.33 Mice were sacrificed up to 30 days after the injections. By the 9th day post-injection, 37 of 38 mice exhibited hydrocephalus, neurological abnormalities and developmental deficits. On the 20th to 23rd day postinjection, approximately 60% developed spontaneous subdural hemorrhages in the parietal and occipital regions. The authors observed organizing subdural blood clots in some cases surrounded by brownish blood clots. The occipital cortex was noted to become membrane-thin and perforated at the occipital horns. Histological observations also confirmed similarities to human CSDH with a thin inner membrane of several cell layers and a thick outer membrane with fibroblasts, neovascularity, hemosiderin-laden macrophages and fresh hemorrhage. The authors hypothesized that the hematomas formed when the occipital horns of the lateral ventricles perforated into the subdural space and the ventricles collapsed, tearing bridging veins and causing subdural bleeding.
Discussion Few studies attempting to model CSDH experimentally have been conducted since 1987, perhaps because existing models have been unsuccessful at replicating this pathology. For these reasons we have reviewed the past and present literature on CSDH, as it is important to understand the strengths and weaknesses of current models so that an effective model can be established in the future. Attempts to create an experimental model of human CSDH have been made in mice, rats, cats, dogs and monkeys. Intracranial studies have been done in all of these species except rats. Injecting various blood products and mixtures into the subcutaneous space of rats, cats and dogs has produced transiently expanding, liquified hematomas in up to half of the animals. The histology of the outer CSDH membrane in humans resembles closely the wall of the hematomas produced by subcutaneous injection of blood and is well-described as the typical response of the body to a hematoma.27 Most hematomas resolved spontaneously. It is well known that chronic expanding hematomas develop in parts of the human body, in the subdural space and even in the brain itself.34,35 The histology of the walls of these lesions is very similar to CSDH. Despite the past studies on CSDH, a reproducible model has not yet been reported. There are a number of limitations with the published models. No reproducible model of a true continually expanding, liquified hematoma that does not resolve spontaneously has been described. Modification of the existing ASDH models may be feasible but rapid resolution of the clot has generally
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occurred in such models, which is also a common problem in CSDH models. Subcutaneous blood injections are of unknown relevance to human CSDH. Whether the pathophysiology of the response to blood in the subcutaneous space is the same as in the subdural space is unknown. The main treatment for CSDH is surgical drainage. Since this leads to faster resolution of human CSDH, it might be the gold standard against which the applicability of an animal model is tested. Since none of the models produces a chronically expanding lesion, the endpoint should have to be faster resolution with drainage compared to without. Head injury precedes CSDH in 60–80% of cases and this is widely believed to be the etiologic factor. However, there are inconsistencies. What is the cause in those with no recollection of trauma? Was the trauma so minor as to be forgotten or are there some other factors? If the first event is tearing of a bridging vein that can occur after such minor injury that it is forgotten, then there should be blood visible on brain imaging right after the injury. However, the senior author has imaged a number of patients where the CT scan is normal right after the injury and a CSDH appears after 2–4 weeks. Finally, a CSDH typically is not near where the bridging veins enter the sinuses, but over the convexity where there are no bridging veins. Therefore, a surgical method aimed at damaging bridging veins may not be a reliable option. Watanabe et al. suggested that fibrin and CSF were essential for creating an expanding, liquified hematoma.23 The continuing expansion of blood plus CSF reported by Watanabe and colleagues was not replicated by Ohshima, who had to treat the dogs with mannitol and heparin.22 However, the findings of Ohshima also have not been replicated. Although Apfelbaum et al. found that CSF was not important for CSDH formation in animal models, human studies have suggested that CSF leakage may be involved in CSDH pathogenesis and recurrence.36 No studies in vitro have examined the role of CSF in clot development. Therefore, further studies must be done to discern the role of CSF in the pathogenesis of CSDH. Brain atrophy is believed to be a predisposing factor to CSDH, given that patients with high cortical atrophy have an increased risk of CSDH. Volumetric analysis of CT brain scans has established an association between the two.37 Cerebral atrophy may facilitate dura–arachnoid separation after trauma and has been shown in cats and dogs to cause fibrininduced proliferation of granulation tissue on the dura.27 The methods used by Watanabe et al., in which a balloon was utilized to create a space for blood injection, mimics the dura–arachnoid separation seen in CSDH patients with cerebral atrophy.23 This model may be especially beneficial for studying the effect of
D’Abbondanza and Macdonald
brain atrophy on CSDH in animals and can lead to a further understanding as to why there is an increased risk for CSDH in older patients with cerebral atrophy. The pathogenesis of CSDH enlargement also has been attributed to various factors including osmotic and oncotic gradients, fibrinolytic activity in the fluid, inflammation, angiogenesis and bleeding from the outer membrane.38 A discussion on the pathogenesis is not the topic of this review, but we suggest that development of an animal model might facilitate understanding of the pathogenesis of human CSDH. The results of this review suggest that this has not been accomplished yet. Studies in the early twentieth century were performed on larger animals, which tended to be used more commonly in experiments compared to now. However, experiments in large animals can be expensive and pose limitations due to insufficient information about and inability to manipulate their genomes. It may be more feasible to first establish a reproducible model in smaller animals, such as rodents, and then attempt CSDH models in larger species. Mouse models are advantageous due to extensive knowledge of their genome, similarity to human physiology, accelerated lifespan, cost effectiveness and ability to manipulate the genome. Rodent models have been widely used to replicating the pathophysiology of other human diseases, such as subarachnoid hemorrhage.39–42 Compared to the subarachnoid space targeted in these studies, the subdural space is relatively easy to access in mice. Thus, rodent models of CSDH may be one avenue to explore initially. A model that replicates the characteristics of human CSDH is essential; this includes a persistent, expanding, liquefied hematoma encased in a neomembrane. Since CSDH is most prevalent in the elderly and males,1,43 future studies may benefit from the use of older male animals. Given that CSDH is a result of an autologous, spontaneous hemorrhage into the potential subdural space, it may be beneficial to use a model that simulates all aspects of CSDH pathogenesis. This may be achieved by utilizing fresh blood injection into the subdural space rather than subdural or subcutaneous clot placement. A model in which bridging veins are weakened followed by a form of mild head trauma may also be a novel method of inducing spontaneous CSDH. Future CSDH research should focus on establishing a reproducible animal model of CSDH as it is essential for improving treatment methods and, ultimately, quality of life for CSDH patients.
Disclosures R. L. Macdonald receives grant support from the Physicians Services Incorporated Foundation,
Experimental Models of Chronic Subdural Hematoma
Brain Aneurysm Foundation, Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada. R. L. Macdonald is a consultant for Actelion Pharmaceuticals and Chief Scientific Officer of Edge Therapeutics, Inc.
Acknowledgements None.
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