Int. I. Radiaiion
Oncology
Bid.
Phys..
1976. Vol.
1, pp. 679485.
Perpamon
Press.
Printed
in the U.S.A.
CHANGES IN PLATELET AND MICROCIRCULATION FUNCTION INDUCED BY IONIZING RADIATION TO THE LIVER HAIM I. BICHER, M.D., Ph.D.,? DONALD W. ASHBROOK, B.S.,* DONALD R. HARRIS, M.D.§ and GLENN V. DALRYMPLE M.D.1 Departments
of Pharmacology and Radiology, University of Arkansas Sciences Campus, Little Rock, AR 72201, U.S.A.
Liver, Mkro&cula tion, Platelet aggregation, IO&@ Fhliath
hepatitis.
INTRODUCTION
radiation, Tissue reoxygenation,
treated and the dosage, central venous thrombosis and congestive hemorrhage may occur. Significant secondary degeneration of the liver may ensue during the latter part of the acute period or thereafter.” Ingold et al.” described the clinical features of radiation hepatitis. The principal manifestations are hepatomegaly and ascites, sometimes accompanied by jaundice and concomitant deterioration of liver function tests. The condition may be transient and may require little therapy, or it may terminate in death from hepatic insufficiency. These authors concluded that (1) histologically, the acute effects of radiation were centrolobular hemorrhage and sinusoidal congestion with adjacent minimal hepatic cellular atrophy. The late or chronic changes were not distinct, but did show more hepatic cellular damage and changes of the central vein walls, and (2) there appeared to be a relationship between increas-
Among the major mechanisms by which radiation affects mammalian tissue are (1) inhibition of the proliferative capacity of rapidly dividing cells, and (2) irreversible damage to elements of the microcirculatory system.18 Organs such as liver, kidney and brain which are not characterized by rapidly proliferating cells, exhibit a relatively pronounced radiosensitivity in spite of a very slow rate of cellular turnover. The sensitivity of the microcirculatory system function to radiation could provide the reason for the radiosensitivity of the entire organ or tissue. After liver irradiation, the acute period tends to be relatively asymptomatic clinically since the hepatic cells are relatively resistant to direct cytocidal actions and since relatively large doses are required to cause marked acute inflammation in the liver. However, depending upon the volume of the liver tAssociate Professor of Pharmacology, Resident in Radiation Therapy. STechnologist, Department of Nuclear Medicine. §Assistant Professor, Radiation Oncology.
for Medical
liPProfessor and Chairman , Department of Radiology; and Veterans Administration Hospital, Little Rock, Arkansas.
679
680
Radiation Oncology 0 Biology 0 Physics
ing doses of irradiation and the incidence of liver changes. Doses of less than 3000rad to the entire liver in 4 weeks seemed relatively safe. The above mentioned facts, namely the chronicity of the “delayed” (more than 6 months after treatment) changes and the vascular nature of the “immediate” (during the first 6 months following treatment) effects of liver irradiation tend to emphasize the possibility that the mechanism of this insult is mediated primarily through microcirculatory system damage. A similar suggestion was advanced in a recent paper by Schultz, Glatstein and Kaplan discussing the role played by platelets in this respect.*’ In previous publications”A4J we have described microcirculatory damage to parenchymatous organs induced by intravascular platelet and red cell aggregation, as these processes tend to interfere with tissue oxygenation. The scope of this investigation was to elucidate the effects of @Co ionizing radiation on liver microcirculation, function and oxygenation. METHODS AND MATERIALS Oxygen determinations The oxygen electrodes used in this series of
experiments consist of a Teflon coated platinum/iiiclium alloy wire (120 ~1 dia., 2 cm long) that is advanced into the liver tissue through the lumen of a 26G needle after the needle has penetrated the liver capsule. The electrode is allowed to “float” with the normal respiratory excursions of the liver and is attached to the instrumentation by a 40 p gold wire which will not hamper this movement. The electrodes are coated with Rhoplex AC 35 (a water carrying, highly adhesive, oxygen previous material, obtained from Rohm & Haas, Philadelphia, Pa.) which is used to delay the “poisoning” of the electrode surface by protein electrophoresis. The reference electrode is composed of a pure silver wire coated with AgCl and encased in an agar-KC1 bridge. This reference electrode is placed subcutaneously or inside the abdominal cavity. The electrode is calibrated in vitro prior to
lW6, Vol. 1, Number 7 and Number 8
insertion into the liver using the technique developed by Silver.” Reoxygenation times (RT), or the period of time required for partial pressure of oxygen in tissue (TpG2) to return to its original level after a 60 set period of anoxic anoxia produced by breathing pure Nz, was determined as previously described.6 This measurement has been found to be a good indicative parameter of the ability of the microcirculation to deliver oxygen to tissue. In each experiment, five determinations were made at four different locations in the liver. Determination aggregation Photoelectric
of platelet
adhesiveness
and
method platelet aggregation.
The photoelectric method to determine platelet aggregation in platelet-rich plasma (PRP) has been described by Born.” Continuously stirred PRP is placed in a transilluminated test tube, and the amount of light transmitted is measured with a photocell. Platelet aggregation induced by adenosine diphosphate (ADP) changes the amount of light received by the photocell and can, thereby, be recorded. In order to determine platelet reactivity to ADP in animals pre or post irradiation, a dose response curve is carried out for a sample from each animal, using increasing ADP concentrations from 0.1 to 10 @ per ml. The ADP concentration giving a “mean” response (i.e. midway between minimum and maximum response) is determined. Rolling tube platelet adhesiveness test. In this method, previously described,’ a volume of 1.0 ml/tube of anti-coagulated blood is placed in a series of 10 ml, 1.2 cm dia. non-siliconized test tubes containing no drug (control), and varying concentrations of ADP. The concentrations are less than, equal to, and more than the amount of ADP required for aggregation as determined by the photoelectric method. After rotation, adhesive platelets adhere to the wall of the test tube, and those remaining in the blood are counted. The differences between the platelet counts of the control and the tubes containing ADP indicate percentages of platelet adhesiveness caused by the drug. The effective dose of ADP to induce an increase in platelet adhesiveness
Microcirculation and liver irradiationOH.
is determined in this manner. A change in the amount of ADP required to induce this effect in an individual post-irradiation animal as compared to that animal’s pre-irradiation requirement indicates the increase in platelet adhesiveness.
Histology,
liver function
tests and
blood
chemistries
Liver tissue samples were obtained by wedge biopsy, and were fixed and stained using standard histological techniques for light and electron microscopy studies. Liver function *tests and blood chemistries including lactic dehydrogenase (LDH), serumglutomic oxaloacetic transaminase @GOT) Bilirubin, Total Cohoid, Serum Albumin, Total Serum Protein, albumen/globulin (A/G Ratio), Prothrombin Time, Alkaline Phosphatase,? HCT, WBC, RBC, BUN, Na’, K’ were performed on samples drawn before and after radiation treatments. Standard clinical methodology was used in all tests.
I. BICHERet al.
681
General experimental plan
The experiments were performed on female beagle dogs, one year old when treatment was started. All determinations were performed prior to radiation, and then again 2 weeks after radiation treatments ended. Whole liver radiation was performed through parallel lateral opposing ports after radiological confirmation of field localization (see Fig. 1). The animals were conscious during treatment, and were immobilized in a sling which was suspended from a frame. Only the port area was exposed through opposing windows in the sling fabric. The animals were treated to 4600 rad delivered in 23 treatments, total treatment time being 35 days. A -Co Teletherapy half value layer (HVL) 11 pb unit at a Skin Source Distance (SSD) of SOcm was used. Isodose curves were determined for each animal. When determining TpOz, the animals were under barbituate anesthesia with tracheal intubation and cannulation of the femoral artery and vein. Blood samples for in vitro
Fig. 1. Lateral verificationport. 8 x 10 cm field placed over the liver region. tHCT = hematocrit; WBC = white blood cell count; RBC = red blood cell count: BUN = blood urea
nitrogen.
Radiation Oncology ??Biology 0 Physics
682
studies were obtained from the cammlated femoral vein. All surgical procedures were performed under sterile conditions. RESULTS At the present time, pre and post irradiation studies have been completed in 9 beagles. Reoxygenation times
It was found that the normal reoxygenation time in the dog’s liver is 2.14 min. This figure is larger than the RT in the cat’s brain (0.68 min) as previously reported.’ After ionizing radiation the liver RT is prolonged significantly to an average of 3.26min (see Table 1, Figs. 2 and 3). It should be noted that individual differences in animal responses tend to be Table 1. Average beagle reoxygenation timesLiver (statisticaltest used was two-tailed t-test) DOS
Pre-irrad.
Post-irrad.
A-5
1.56f 0.42 2.53 + 1.35 1.30 * 0.20 2.00+ 1.13 l&I+-0.49 2.72 = 1.54 2.80 + 0.84 3.26 +-0.88 1.58 z 0.21 2.272 1.15
3.36-c 1.11 4.87 r 1.21 5.500 1.89 1.84kO.45 2.72 2 0.65 2.17k0.71 3.15-to.90 3.79 t: 0.92 2.98 2 0.93 3.35 -c 1.49
A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 Av.
p vaiues
-Co.001 CO.001 CO.001 CO.7
Oncology
Bid.
Phys..
1976. Vol.
1, pp. 679485.
Perpamon
Press.
Printed
in the U.S.A.
CHANGES IN PLATELET AND MICROCIRCULATION FUNCTION INDUCED BY IONIZING RADIATION TO THE LIVER HAIM I. BICHER, M.D., Ph.D.,? DONALD W. ASHBROOK, B.S.,* DONALD R. HARRIS, M.D.§ and GLENN V. DALRYMPLE M.D.1 Departments
of Pharmacology and Radiology, University of Arkansas Sciences Campus, Little Rock, AR 72201, U.S.A.
Liver, Mkro&cula tion, Platelet aggregation, IO&@ Fhliath
hepatitis.
INTRODUCTION
radiation, Tissue reoxygenation,
treated and the dosage, central venous thrombosis and congestive hemorrhage may occur. Significant secondary degeneration of the liver may ensue during the latter part of the acute period or thereafter.” Ingold et al.” described the clinical features of radiation hepatitis. The principal manifestations are hepatomegaly and ascites, sometimes accompanied by jaundice and concomitant deterioration of liver function tests. The condition may be transient and may require little therapy, or it may terminate in death from hepatic insufficiency. These authors concluded that (1) histologically, the acute effects of radiation were centrolobular hemorrhage and sinusoidal congestion with adjacent minimal hepatic cellular atrophy. The late or chronic changes were not distinct, but did show more hepatic cellular damage and changes of the central vein walls, and (2) there appeared to be a relationship between increas-
Among the major mechanisms by which radiation affects mammalian tissue are (1) inhibition of the proliferative capacity of rapidly dividing cells, and (2) irreversible damage to elements of the microcirculatory system.18 Organs such as liver, kidney and brain which are not characterized by rapidly proliferating cells, exhibit a relatively pronounced radiosensitivity in spite of a very slow rate of cellular turnover. The sensitivity of the microcirculatory system function to radiation could provide the reason for the radiosensitivity of the entire organ or tissue. After liver irradiation, the acute period tends to be relatively asymptomatic clinically since the hepatic cells are relatively resistant to direct cytocidal actions and since relatively large doses are required to cause marked acute inflammation in the liver. However, depending upon the volume of the liver tAssociate Professor of Pharmacology, Resident in Radiation Therapy. STechnologist, Department of Nuclear Medicine. §Assistant Professor, Radiation Oncology.
for Medical
liPProfessor and Chairman , Department of Radiology; and Veterans Administration Hospital, Little Rock, Arkansas.
679
680
Radiation Oncology 0 Biology 0 Physics
ing doses of irradiation and the incidence of liver changes. Doses of less than 3000rad to the entire liver in 4 weeks seemed relatively safe. The above mentioned facts, namely the chronicity of the “delayed” (more than 6 months after treatment) changes and the vascular nature of the “immediate” (during the first 6 months following treatment) effects of liver irradiation tend to emphasize the possibility that the mechanism of this insult is mediated primarily through microcirculatory system damage. A similar suggestion was advanced in a recent paper by Schultz, Glatstein and Kaplan discussing the role played by platelets in this respect.*’ In previous publications”A4J we have described microcirculatory damage to parenchymatous organs induced by intravascular platelet and red cell aggregation, as these processes tend to interfere with tissue oxygenation. The scope of this investigation was to elucidate the effects of @Co ionizing radiation on liver microcirculation, function and oxygenation. METHODS AND MATERIALS Oxygen determinations The oxygen electrodes used in this series of
experiments consist of a Teflon coated platinum/iiiclium alloy wire (120 ~1 dia., 2 cm long) that is advanced into the liver tissue through the lumen of a 26G needle after the needle has penetrated the liver capsule. The electrode is allowed to “float” with the normal respiratory excursions of the liver and is attached to the instrumentation by a 40 p gold wire which will not hamper this movement. The electrodes are coated with Rhoplex AC 35 (a water carrying, highly adhesive, oxygen previous material, obtained from Rohm & Haas, Philadelphia, Pa.) which is used to delay the “poisoning” of the electrode surface by protein electrophoresis. The reference electrode is composed of a pure silver wire coated with AgCl and encased in an agar-KC1 bridge. This reference electrode is placed subcutaneously or inside the abdominal cavity. The electrode is calibrated in vitro prior to
lW6, Vol. 1, Number 7 and Number 8
insertion into the liver using the technique developed by Silver.” Reoxygenation times (RT), or the period of time required for partial pressure of oxygen in tissue (TpG2) to return to its original level after a 60 set period of anoxic anoxia produced by breathing pure Nz, was determined as previously described.6 This measurement has been found to be a good indicative parameter of the ability of the microcirculation to deliver oxygen to tissue. In each experiment, five determinations were made at four different locations in the liver. Determination aggregation Photoelectric
of platelet
adhesiveness
and
method platelet aggregation.
The photoelectric method to determine platelet aggregation in platelet-rich plasma (PRP) has been described by Born.” Continuously stirred PRP is placed in a transilluminated test tube, and the amount of light transmitted is measured with a photocell. Platelet aggregation induced by adenosine diphosphate (ADP) changes the amount of light received by the photocell and can, thereby, be recorded. In order to determine platelet reactivity to ADP in animals pre or post irradiation, a dose response curve is carried out for a sample from each animal, using increasing ADP concentrations from 0.1 to 10 @ per ml. The ADP concentration giving a “mean” response (i.e. midway between minimum and maximum response) is determined. Rolling tube platelet adhesiveness test. In this method, previously described,’ a volume of 1.0 ml/tube of anti-coagulated blood is placed in a series of 10 ml, 1.2 cm dia. non-siliconized test tubes containing no drug (control), and varying concentrations of ADP. The concentrations are less than, equal to, and more than the amount of ADP required for aggregation as determined by the photoelectric method. After rotation, adhesive platelets adhere to the wall of the test tube, and those remaining in the blood are counted. The differences between the platelet counts of the control and the tubes containing ADP indicate percentages of platelet adhesiveness caused by the drug. The effective dose of ADP to induce an increase in platelet adhesiveness
Microcirculation and liver irradiationOH.
is determined in this manner. A change in the amount of ADP required to induce this effect in an individual post-irradiation animal as compared to that animal’s pre-irradiation requirement indicates the increase in platelet adhesiveness.
Histology,
liver function
tests and
blood
chemistries
Liver tissue samples were obtained by wedge biopsy, and were fixed and stained using standard histological techniques for light and electron microscopy studies. Liver function *tests and blood chemistries including lactic dehydrogenase (LDH), serumglutomic oxaloacetic transaminase @GOT) Bilirubin, Total Cohoid, Serum Albumin, Total Serum Protein, albumen/globulin (A/G Ratio), Prothrombin Time, Alkaline Phosphatase,? HCT, WBC, RBC, BUN, Na’, K’ were performed on samples drawn before and after radiation treatments. Standard clinical methodology was used in all tests.
I. BICHERet al.
681
General experimental plan
The experiments were performed on female beagle dogs, one year old when treatment was started. All determinations were performed prior to radiation, and then again 2 weeks after radiation treatments ended. Whole liver radiation was performed through parallel lateral opposing ports after radiological confirmation of field localization (see Fig. 1). The animals were conscious during treatment, and were immobilized in a sling which was suspended from a frame. Only the port area was exposed through opposing windows in the sling fabric. The animals were treated to 4600 rad delivered in 23 treatments, total treatment time being 35 days. A -Co Teletherapy half value layer (HVL) 11 pb unit at a Skin Source Distance (SSD) of SOcm was used. Isodose curves were determined for each animal. When determining TpOz, the animals were under barbituate anesthesia with tracheal intubation and cannulation of the femoral artery and vein. Blood samples for in vitro
Fig. 1. Lateral verificationport. 8 x 10 cm field placed over the liver region. tHCT = hematocrit; WBC = white blood cell count; RBC = red blood cell count: BUN = blood urea
nitrogen.
Radiation Oncology ??Biology 0 Physics
682
studies were obtained from the cammlated femoral vein. All surgical procedures were performed under sterile conditions. RESULTS At the present time, pre and post irradiation studies have been completed in 9 beagles. Reoxygenation times
It was found that the normal reoxygenation time in the dog’s liver is 2.14 min. This figure is larger than the RT in the cat’s brain (0.68 min) as previously reported.’ After ionizing radiation the liver RT is prolonged significantly to an average of 3.26min (see Table 1, Figs. 2 and 3). It should be noted that individual differences in animal responses tend to be Table 1. Average beagle reoxygenation timesLiver (statisticaltest used was two-tailed t-test) DOS
Pre-irrad.
Post-irrad.
A-5
1.56f 0.42 2.53 + 1.35 1.30 * 0.20 2.00+ 1.13 l&I+-0.49 2.72 = 1.54 2.80 + 0.84 3.26 +-0.88 1.58 z 0.21 2.272 1.15
3.36-c 1.11 4.87 r 1.21 5.500 1.89 1.84kO.45 2.72 2 0.65 2.17k0.71 3.15-to.90 3.79 t: 0.92 2.98 2 0.93 3.35 -c 1.49
A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 Av.
p vaiues
-Co.001 CO.001 CO.001 CO.7
