The permeability of capillaries in rabbit muscle spindles, intramuscular nerves, and extrafusal muscle to intraaortically infused horseradish peroxidase (HRP) was studied. The permeabilityof the capsule cells of the muscle spindles was also examined. Immediately following the infusion of HRP, most extrafusal capillaries contained a dense accumulation of HRP reaction product, which began to escape into the surrounding extracellular space within 2-3 min. In contrast, the concentration of HRP in the intrafusal and endoneurial capillaries was much lower than that in extrafusal capillaries, and HRP was never found outside the former vessels. In additicn, HRP never completely penetrated through the spindle capsule from the extracellular into the periaxial space, although it was regularly found between the outer two or three layers of capsule cells. The results indicate that the permeability characteristics of the muscle spindle capillaries and capsule to the tracer protein HRP are very similar to those of capillaries in peripheral nerve. MUSCLE & NERVE
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1979
PERMEABILITY OF MUSCLE SPINDLE CAPILLARIES AND CAPSULE WILLIAM R. KENNEDY, MD, and KWON S. YOON, MS
It is widely accepted that the blood-brain barrier plays an important role in maintaining envirorimental stability within the ccntral nervous system.14A similar barrier is considered to exist in the peripheral nervous system, where it has been shown in both rats and mice that the endoneurial capill a r i e ~ ' ~and ~ ' ~the * ~p~e r i n e ~ r i u r n ' ~ ,prevent ' ~ * ~ ~ certain tracer substances from entcring into the endoneurial space. It is known that large-molecularweight substances penetrate the barrier in some experimental pathologic conditions and also penetrate through natural openings at the dorsal root entry zone17325 and probably at motor ending^.^ T h e blood -nervous tissue barrier of brain and peripheral nerve is almost certainly a product of
From the Department of Neurology, University of Minnesota Hospitals Minneapolis MN Acknowledgments The authors wish to thank Or Donald C Quick for his valuable suggestions This study was supported by National Institutes of Health grant no NS 10969 and by a grant from the American Diabetes Association. Minnesota Chapter Address reprint requests to Or Kennedy at the University of Minnesota, Box 187-Mayo University of Minnesota Hospitals. Minneapolis, MN 55455 Received for publication October 12, 1978 revised manuscript accepted
for publication December 18, 1978 0148-639X/0202/0101$00 OO/O 1979 Houghton Mifflin Professional Publishers @
Muscle Spindle Capillaries
specialized structural and functional characteristics of the capillary endothelial cells, T h e tight junctions in the iritercellular clefts of endothelial cells in brain3 and nerve capillaries'* limit the outward diffusion of materials that normally occurs from capillaries in muscle and in some other tissues. There appears to be virtually no pinocytotic transfer of larger molecules, including proteins, across the endothelial cell membranes. Instead, effective passage depends on the presence of suhstance-specific carrier systems o r bears a direct relationship to the lipid solubility of the entering molec~iles.'~ We felt it of interest to investigate whether the above barriers between blood, the endoneurial compartment, and the extraperineurial spaces extend peripherally into the sensory organ-the muscle spindles. T h e capillaries of the muscle spindle are of special interest because they are identical in size and character to capillaries in peripheral nerves," and because their arterioles a n d venules of origin provide a more direct circulation than d o those of extrafusal m ~ s c l e .These '~ capillaries might thus provide a model for the study of some features of the blood -nervous system barrier. T h e present study compares the permeability of intrafusal (IF) capillaries and intramuscular endoneurial capillaries with that of extrafusal (EF)
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capillaries of skeletal muscle using the tracer protein horseradish peroxidase (HRP). Also assesed is the permeability to HRP of the perineurium-like capsule of the spindle. Rabbit spindles from tenuissimus muscle were chosen for study because they consistently contain one or more capillaries inside the periaxial space.2 MATERIALS AND METHODS
Forty New Zealand white rabbits weighing from 1 to 3 lbs (2.2-6.6 kg) received HRP injections or served as controls. HRP (Type 11, RZ 1.5) was obtained from Sigma Laboratories, St. L.ouis, MO. Six rabbits were initially tested for hypersensitivity to HKP. After inhalation anesthesia with methoxyflurane (Penthrane, Abbott Laboratories, North Chicago, IL), the fur on the abdomen was clipped short, arid 6 in1 of Evans blue dye was injected intravenously. Immediately following this, 5, 50, and 100 mg of HRP in 0.1-ml aliquots of saline were in-jected intradermally on the abdomen for comparison with similar 0.1-ml injections of saline alone and of saline containing 10 or 100 mg of histamine. Approximately 30 min following the intradermal injections, the maximum diameter of the blue wheal was measured.
Sensitivity Testing.
Capillary Permeability to HRP. T h e rabbits
that were
to receive intravascular HRP were anesthetized as
described above; the fur was then removed from the abdomen and from both hind extremities. Next, both tenuissimus muscles were exposed over most of their length. The otherwise-undisturbed muscles were then covered again by adjacent muscle and skin. A heating lamp and a warming pad were used to help maintain body temperature. T h e abdominal aorta was then exposed and injected with either 100 or 400 mg of HKP per kilogram body weight in 1 ml of isotonic saline. At predetermined intervals between 1 and 40 min postinjection, a tenuissimus muscle was removed and pinned at a slightly stretched length in 5% glutaraldehyde buffered by 0.1 M sodium cacodylate, pH 7 . 3 , for 2 hr at room temperature. After washing in the same buffer, strips of EF muscle overlying muscle spindles were carefully microdissected free until the spindle capsule was exposed. T h e entire muscle spindle, with the underlying undisturbed bed of EF muscle and the adjacent nerve, was then removed en bloc. The specimens were then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB) to which fresh hydrogen peroxide had been added.'l I n some specimens, the spindle cap-
102
Muscle Spindle Capillaries
sule was opened to ensure better exposure of I F capillaries to DAB. T h e 5pecimens were postfixed in 2% osmium tetroxide for 2 hr, after which they were dehydrated by passage through graded ethanol and embedded in Epon. Ultrathin sections were cut on an LKB Ultratome I11 and were examined electron microscopically. Some sections were stained on the grid with saturated uranyl acetate and lead citrate, but most were left unstained so that the distribution of the reaction product could be more accurately defined. Spindles from non-HKP-injected animals were treated in an identical manner: this included incubation in DAB. Capsule Permeability to HRP. I n a few rabbits that had not been injected with HRP, a search was made of the tenuissimus muscle in situ for living muscle spindles; the dissecting microscope was used for this purpose. When a spindle was located, most of the overlying EF fibers were carefully removed. Spindles exposed in this manner retain their tonic and phasic reactivity to muscle stretch, as determined by in-vivo recording from the intramuscular nerve. The spindle and the adjacent EF muscle were then periodically flooded by a solution containing 1 mg of HRP per milliliter to test for permeability of' the spindle capsule to the externally applied tracer. The spindles were removed at varying intervals up to 45 rnin, at which point they were fEed and processed as indicated above. RESULTS
The diameters of Ebans blue stain around the injection sites of saline and 5 mg HRP were equa,l and never exceeded 2.5 mm. In four of the six animals that had been tested for sensitivity, the stain at the 50-mg HKP site was also less than 2.5 mm, while in the other two animals it averaged 5.5 mm. The stain at the 10-mg histamine site averaged 11 mm. It was concluded that the rabbit had minimal sensitivity to Sigma 'l'ype I1 IHRP. We failed t o detect any clinical manifestation of hypersensitivity to the intraaortic injections of HRP, even in quantities of 400 mg/kg. There were no fatalities to anesthesia. Sensitivity to HRP.
Preparation of transverse sections of the processed tissue blocks provided a convenient means of comparing capillaries in the periaxial space of the spindle with those between adjacent EF muscle fibers (fig. 1A). It immediately became evident that the former were larger in diameter and in area. Higher magnification of ultrathin sections (fig. 1B) showed that the typical IF capillary Controls.
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Figure I . (A) Transverse section of a spindle and surrounding extrafusal (EF) muscle, showing two periaxial capillaries (black arrowheads) and EF capillaries (arrows). Other structures of the spindle are the outer capsule (OC), periaxial space (psj, inner capsule (IC), myelinated nerve (N), nuclear bag fiber (nb), nuclear chain fiber (nc), and sensory terminals (St). Phase contrast, bar = 25 pm. (B) Transverse section of an intrafusal capillary in the periaxial space illustrates overlapping region of encircling fibrocytic processes (arrowhead), longitudinal coliagen bundles (col), multilayered basement membrane (bm), enclosing endothelial cells and pericytic processes (pc). and part of a red blood corpuscle (rbc). Treated with tannic acid en bloc; stained with lead citrate and uranyi acetate on the grid. Bar = I pm.
was ensheathed by thin fibrocyte-like processes which were joined by tight junctions where two processes overlapped. Small bundles of longitudinal collagen fibers were situated under the sheath. Collagen also encircled the capillary. 'I'he basement membranes of the endothelial cells were often multilayered. T h e membranes usually divided to surround the many pericytic processes that heavily invested the endothelial cells. Vesicles within the endothelial cells were small and few.*' Following intraaortic injection of HRP, all vessels in the outer portion of the muscle block were filled with the dark reaction product. Central vessels were not demonstrated to be uniformly filled because the DAB failed to penetrate fully into the central core of the larger specimens. EF vessels were in varying stages of patency
Capillary Permeability.
Muscle Spindle Capillaries
or collapse. In the specimens that had been placed in fixative 60 and 90 sec postinjection, the reaction product was entirely contained within the capillary. .4t that time, adluminal pinocytotic vesicles were fdlcd (fig. 2). ,4t slightly later intervals, the cytoplasmic and then the abluminal vesicles filled. T h e reaction product first appeared outside the capillary in some of thc specimcns that had been placed in fixative 2 rnin after the start of the HRP injection. I n specimens removed at 3 min, some of the reaction product was always found in the extraccllular space (fig. 3A). We did not attempt to establish more precisely the time required for HRP t o cscape from the EF capillary, because the fixation time could not be determined. Simionescu et alZ3determined fixation time for rat cremasteric musclc, using this value as an estimate for fixation time of mouse diaphragm. When the cremasteric
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greater dimension than those in EF muscle (fig. LA). 111 some IF capillaries, portions of the cytoplasm of the cndothelial cells were darkly stained (fig. 3Rj. I n the preparations whcre the capsule had been opened to allow lor better penetration of the DAB into the spindle, the appearance did not differ from that described above fbr the intact spindle. In these experiments, the HRP solution was flooded onto t h e living spindle lying in situ in the E F bed. HRP reactioii- product was found to have penetrated between individual EF muscle iibers arid into the EF capillaries. 'The appearance was similar to that of the capillary studied after intraarterial injection. T h e intracellular clefts and the pinocytotic vesicles, particularly those on the abluniirial surface, were filled (fig. 5A). H RP reaction product surrounded the spindle capsule but did not penetrate into the periaxial region, even after 45 rnin of exposure. In most instances, HKP reaction product w a s found in t h e space between the outer two or three layers of capsule cells (fig. 58). 'l'hese spaces were often wider than the more internal iionpenetrated perineurial cell layers. Only a small proportion of the pinocytotic wsicles of capsule cells were filled with reaction product. H RP reaction product was found adjacent to the I F muscle fibers at the distal pole of only one spindle. 'I'his specimen had an obvious mechanical disruption of the outer capsule layer. Intrafusally, thc HRP coricentration was higher under the area of disruption but was absent in the region opposite the disruption. Additional observations of capsule permeability to HRP were made from spindles removed from rabbits that had been injected intraaortically with HRP. In these animals, the HKP that escaped from EF capillaries migrated to the spindle and bathed the capsule for periods of up to 45 min. T h e results were identical to those obtained from spindles flooded with HRP as recorded above: HRP was found between the outer two to three capsule cell layers but never within the periaxial space.
Capsule Permeability.
Figure 2 Extrafusal (EF) capillary at 1 min postinjection HRP reaction product ( r p ) IS in adluminal vesicles and in luminal extent of the interceliular deft but not in extracellular space Unstained, bar = 7 pm
preparation was covered with a parafor-maldehyde-glutaraldehyde fixative, the circulation of red blood corpuscles ceased in 25 sec. Fixation of other preparations may require longer intervals. In other studies (W.R. Kennedy, unpublished data) we have noted, while recording from the sensory nerve of a fully exposed muscle spindle, that the action potentials comprising the sensory response to stretch continued for approximately 2.5 min following direct flooding with a 5% glutaraldehyde solution. It was not our intention in these experimciits to establish the timing or route of escape of' HRP from EF vessels. HRP reaction product was also consistently present in the capillaries of the muscle spindles and in the endoneurial capillaries of the central nerve in the tenuissimus muscle. The reaction product appeared flocculent and was obviously of lower concentration than that found in the EF capillaries (figs. 3B and 4). Reaction product never appeared within the periaxial space outside IF capillaries; nor did it appear outside the endoneurial capillaries, even when the interval following injection was 40 min, although reaction product could be demonstrated within the lumina of these vessels at all times. All capillaries within the spindle and within the nerve were widely patent. The vesicles in the capillaries were only rarely found to contain HRP reaction product. As determined previously, capillaries from the spindle and nerves were of
104
Muscle Spindle Capillaries
DISCUSSION
T h e permeability of blood vessels in muscle spindles has not been previously investigated. Only recently was the microcirculation to spindles found to be separate from that t o the surrounding EF muscle.13Artenoles to spindles are third- to f-ourthorder branches from the central muscle artery, whereas most arterioles to EF capillaries are sixth-
MUSCLE & NERVE
Mar/Apr 1979
Figure 3 [A) Extrafusai (EF) capillary 3 min postinjection HRP reaction product (rp) has leaked out into surrounding fissue Unstained, bar = 7 p.m (6') intrafusai capiliary 3 rnin postinjection HRP reaction product (rp) remains within the iumen, none has leaked into the periaxial space (psi Unstained, bar = 7 pn
t o eighth-order branches. T h e final arteriolc ends
in two or three capillaries going only to the spindle. These capillaries d o not anastomose wit.h capillaries in EF muscle. I n addition, spindle capillaries were discovered to be structurally different from muscle capillaries." Spindle capillaries are larger than EF capillaries because of a greater endothelial cell complement. T h e clefts between the endothelial cells are consistently found t o have tight junctions, arid the vesicles within thc cells are fewer. Pericyte and basement rnenibraric coverage is heavier. Most of these features are shared with capillaries of peripheral nerve and brain. In addition, the spindle, like nerve, has a perineurialcell-like barrier. separating it from the surrounding extracellular space." These similarities raise the expectation that the permeability 01' IF vessels might be similar to that of vessels in the endoneurium and in the central nervous system. For example, certain dyes and proteins that escape frorn epirieurial blood vesscls remain inside endoneurial vessels of'the rat and mouse.I6 There is species variation, however, for these substances leak out of endo-
Muscle Spindle Capillaries
neurial capillaries of the chicken and guinea pig.'6325 Vessels of the dorsal root ganglion are permeable in all specime~is."'~~ Olssonl' also found some leakage of Evans blue conjugated t o albumin from endoneurial capillaries of the rabbit. I t is well known that horseradish peroxidase and lanthanum are prevented from leaking out of capillaries in -the brain and peripheral nerves by tight junctions between adjacent endothelial cell^.^,'^*^^ T h e results of the present experiments extend this observation to capillaries within t.he muscle spindle, and extend the findings of Olssori and KeeseI8 to include endoneurial capillaries of intramuscular nerve. The floccular nature of the reaction product in IF and endoneurial capillaries suggests that these vessels contain a lower concentration of HRP than do EF capillaries. This difference was just as evident when 100 mgikg was injected as it was with the 400-mgikg infusion. It can be logically argued that HRP might leak out of IF and endoneurial capillaries if the concentrat.ion were further increased. At the high concentration of 400 mg/kg
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Figure 4 Endoneurial capillary 3 min postinjection HRP reaction product (rp) remains within the lumen Unstained, bar = 1 pni
used in these studies, one would expect some leakage or a more prominent presence of HRP in endothelial cell vesicles or intercellular clefts if permeability were possible. The absence of such h i d ings is evidence of'a difference in permeability that serves as a functional barrier for IF capillaries. The finding of tight junctions between adjacent endothelial cells of IF capillaries would appear to corroborate our conclusions regarding the presence of a tight barrier to HRP. The blood pressure and blood flow ineasurements of Fronek and Zweifach,x performed in EF blood vessels of varying size and distance from the main arteriole in cat tenuissirnus muscle, have led us to suspect that blood flow and pressure are high in spindle capillaries as compared to EY capillaries. The large I F capillaries arise directly from thirdorder arterioles, whereas the smaller EF capillaries arise from sixth- to eighth-order arterioles.'3 The apparently lower concentration of HRP in these vessels, suggested by the floccular nature of the reaction product, would be compatible 5vit.h high blood How. Studies of vessels in the inner ear following in-
106
Muscle Spindle Capillaries
travasciilar infusion of HRP provide an analogy to our observations regarding IF capillaries. HKP does not leak out of vessels of the basilar membrane, the spiral ligament, 01- the spiral prominence.6~26 In these capillaries, HRP reaction product appeared to be of low concentration and had the same fiocculilr appearance seen in spindle capillaries.6 In the vessels of the adjacent stria vascularis, reaction product filled the lumen as a dense accumulation, also crossing the endothelial cell walls by vesicular transport to extracellular space.6The areas bathed by the capillaries in the basilar membrane, the spiral ligament, and the spiral prominence contain nerve tissue. By inspection, these vessels have the same morphologic f-eatures described herein for spindle and nerve capillaries. In this respect, the observations that blood flow in the spiral ligament was faster than that in the vessels of the stria vascularis of the cochlea20 may be relevant to blood flow in the spindle. Periaxial capillaries are commonly found in humans as well as in cats (personal observations), rabbits,2 and chickens,1s arid are very occasionally found in rats.1° I n these species, capillaries that are presumed to be entering the spindle often run initially for varying distances between layers of capsule cells. Capillaries between the innermost layers in rat and rabbit spindles retain HRP, but those between outer layers leak IIRP (personal observations). Spindles of some smaller species-e.g., rats,'O mice,7 hamsters (personal observation), and snakesg-are nourished by capillaries between layers of capsule cells or by capillaries completely outside the spindle. In the latter spindles, it is presumed that the capsule cells regulate passage of materials from the outside vascular supply to the I F muscle and nerves. Therefore, it would not be too surprising if capsule permeability were found to vary from species to species. The peririeurial cells have generally been accepted as forming a diffusion barrier between extracellular space and eiidoneurium or periaxial space." Electron-microscopic studies have shown that the perineurium prevents penetration of Kerritin4 and €IRP" into the endoneurium. This was confirmed in the present study, where it was dcmonstrated that the inner layers of peririeurial cells of the spindle capsule are a barrier to I I R P . Tracer did enter between the outer two or three cell layers. Olsson and ReeseIxmade the same observation for nerve perineuriuni, but Waggener et alz4found that ferritin did 1101pass even the outer cell layer. It was suggested that the smaller HRP molecule could have passed between incomplete outer layers
MUSCLE 8 NERVE
MariApr 1979
Figure 5 (A) Muscle soaked in HRP for 45 min HRP (arrowhead) has entered the extrafusal (EF) capillary from extracellular space Red blood corpuscle (rbc), reaction (rp) also indicated Unstained. bar = I pni (5) Spmdle soaked i n HRP for 45 rnin HRP is between the outer few capsule layers but dld not penetrate fnto the penaxfal space lntrafusal muscle fiber (‘lF-m), intrafusal capillary (if-cj, outer capsule (OC), and reaction product (rp) are also indicated Lead otrate stained, bar = 7 ~ . c m
of capsule cells.’* We found the outer layer continiious in the few observations made of transverse sect.ions of the entire spindle capsule diameter. It is possible that HRP entered between the outer capsular layers after leaking out of capillaries between the same layers. Neither Olsson and Keese” nor we could est.imate the extent to which HRP underwent vesicular transpor-t across cell layers, but it may be limited, since only a few vesicles contained peroxidatic activity. Another possible pathway by which HRP could penetrate the outer layers of the spindle and nerve perineurial cell layers is t.hrough the clefts of adjacent cells. This would occur if the clefts became more “tight,” progressing from outer to inner capsule layers. A comparison by the freeze-fracture methods of tight junctions in different cell layers would help clarify this possibility, for a relationship does seein to exist between the number of linear strands per zonula occludens and the leakiness of various epithe1ia.j Although Akert et all have demonstrated that the perineurial sealing is of an in-
Muscle Spindle Capillaries
termediate type between “very leaky” and “very tight,” a gradation between cell layers was not mentioned. In summary, t h e permeability characteristics of the capillaries and the capsule of the muscle spindle, as indicated by the use of the tracer protein IIRP, are very similar to those of peripheral nerve. ‘I‘his indicates to us that the normal function of’intrafusal muscle fibers, as well as their sensory arid motor innervation, requires a special intrafusal environment that probably closely resembles the endoneurial environment of the peripheral nerve, these two spaces being in direct continuity. W’e suggest that additional information about the bloodnervous tissue barrier can be obtained by study of the circulation of muscle spindles.
REFERENCES I . Akei-t K, Sandri (:, Weiberl ER, Peper- K , Moor H : T h c finc s t r i i ~ t u r e of the perinellrial epItheliurn. Crll Tissi~rRPS 16.5281 -29.5, 1976.
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2. Banks RW, James N T : T h e blood supply of' rabbit muscle spind1es.j Anal 114:7-12, 1973. 3. Brightman MW, Reese TS: Junctions between intimately apposed cell membranes in the vertebrate brain.] Ce/l Biol 4O:648 -675, 1969. 4. Burke1 CV: The histological fine structure of perineurium. A I C ~Rec L ~ 158:177-189, 1967. 5. Claude P, C;rodenongh DA: Fr-acture faces ot 7onulae occhdentes fkom "tight" and "leaky" epithelia. J Cell Bzol 58:390-400, 1973. 6. D u ~ a l lAJ, Quick CA, Sunderland CR: Horseradish peroxidase in the lateral cochleai- ~ v a l l . A n hOto/~uyigol93:304 316, 1971. 7. Edrvai-ds KY: An ultra~tructur-alstudy of neiiromi~scular spindles in normal mice.,/ Anmt 120:149- 168, 1975. 8. Fronek K , Zweifach BW: Microvascular blood flow in cat tcnuissimus muscle. ~ M i c r o z ~ eR . ~ c~ 12:181 A -189, 1977. 9. Fukami Y. Hunt CC: Structure of snake muscle spindles. J ,Vrurophy+d 33:9-27, 1970. 10. James N T , .Meek GA: T h e blood supply d r a t muscle spindlcs.,/Anat 110:164-165. 1971. 11. Karnovsky MJ: T h e ultrastructure basis of capillaq pel-meability studied with peroxidase as a tracer.J Cell B i d 35213 236, 1967. 12. hliyoshi T, Kennedy WR: Morphometric comparison of capillaries in muscle spindle, nerve. and muscle. Arch N e w roc, i n press. 13. Miyoshi T, Krnnedy WK: Microvasculature of rabbit muscle spindles. Arch Seurol, in press. 14. @I$endorf W H : Permeability of the blood-brain barrier. I n Tower DB (Editor): The ,\'~~~oousSy~tem,Vol 1. New York, Raven Press, 1975, pp 279-289. 15. Olsson Y : Studies on vascular permeability in peripheral nerves. I. Distribution of circulating fluorescent serum albumin i n normal, crushed, sectioned peripheral nerve. Arta
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Nrurtrpnthol (Berlin) 7 : l -15, 1 W i . 16. Olsson Y:Phylogenetic variations in the vascular pernleability of peripheral i i e r ~ e st o serum albumin. ,.lctn Pathol .Microhid Scund 69:621 -623, 1967. 17. Olsson Y:Studies on vascular permeability in peripheral rienes. .4cta lVeuropatho/ (Berlin) L5:l 14-126, 1971. 18. Olsson Y , Rcese '13: Permeability of vasa nervoriim and perineurium in mniisr sciatic nen'e studied hy fluorescence and electron microscopy. ,/ Nrwopntlzo/ E x p Nr71rd 30: I05 119, 1971. spindle cap19. Ovalle \.t'K: Fine striicture of the avia~~-muscle sule. Cell Tivsue Rrs 166:285-298. 1976.
20. Perliman HG, Kumura KS: Observations of the living blood vesels 01. the cochlea. Ann Otol Rhino/ Laqvzgol 64: 1176 1192, 1955. 21. Reese TS, Karnovsky MJ: I h e structural localiLation o f a b h d - b r d i I l harrier tn exogenous peroxidase. J Cell Z3zol 34:244-266, 1967.
22. Shanta TR, Bourne GH: T h e perineurial epithelium-a new concept. I n Bourne GH (Editor): T h f Sfntcturr crnd Funclion uf h e N e w o u c T i ~ s u r ,Vol 1. N e w York, Academic Press. 1968, pp 279-459. 23. Siriiionescu M. Sirriionescu N, Palade CE: Morphometric data on the endotheliurn of blood capillaries. J Cell B i d 60:128-132, 1974.
24. Waggerier JD, Bunri SH, Beggs J: T h e diffusion of t'erritin within the peripheral nelve sheath. An clectron microscopy stiidy.,] Nruropnthol k:xp Nvirrol 24:430-443, 1965. 25, Waksman HH: Experimental study of diphthcric polyneuritis in the rabbit and guinea pig. 111. T h e blood-nerve barrier in the 1abbit.j iVeuroputho1 Exp Neicrol 20:35-77, 1961. 26. Winther PP: l ' h e permeability of the guinea pig cochlear capillaries to horseradish peroxidase. Z %e/!/orsch 114:193 202, 1971.
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1979
PERMEABILITY OF MUSCLE SPINDLE CAPILLARIES AND CAPSULE WILLIAM R. KENNEDY, MD, and KWON S. YOON, MS
It is widely accepted that the blood-brain barrier plays an important role in maintaining envirorimental stability within the ccntral nervous system.14A similar barrier is considered to exist in the peripheral nervous system, where it has been shown in both rats and mice that the endoneurial capill a r i e ~ ' ~and ~ ' ~the * ~p~e r i n e ~ r i u r n ' ~ ,prevent ' ~ * ~ ~ certain tracer substances from entcring into the endoneurial space. It is known that large-molecularweight substances penetrate the barrier in some experimental pathologic conditions and also penetrate through natural openings at the dorsal root entry zone17325 and probably at motor ending^.^ T h e blood -nervous tissue barrier of brain and peripheral nerve is almost certainly a product of
From the Department of Neurology, University of Minnesota Hospitals Minneapolis MN Acknowledgments The authors wish to thank Or Donald C Quick for his valuable suggestions This study was supported by National Institutes of Health grant no NS 10969 and by a grant from the American Diabetes Association. Minnesota Chapter Address reprint requests to Or Kennedy at the University of Minnesota, Box 187-Mayo University of Minnesota Hospitals. Minneapolis, MN 55455 Received for publication October 12, 1978 revised manuscript accepted
for publication December 18, 1978 0148-639X/0202/0101$00 OO/O 1979 Houghton Mifflin Professional Publishers @
Muscle Spindle Capillaries
specialized structural and functional characteristics of the capillary endothelial cells, T h e tight junctions in the iritercellular clefts of endothelial cells in brain3 and nerve capillaries'* limit the outward diffusion of materials that normally occurs from capillaries in muscle and in some other tissues. There appears to be virtually no pinocytotic transfer of larger molecules, including proteins, across the endothelial cell membranes. Instead, effective passage depends on the presence of suhstance-specific carrier systems o r bears a direct relationship to the lipid solubility of the entering molec~iles.'~ We felt it of interest to investigate whether the above barriers between blood, the endoneurial compartment, and the extraperineurial spaces extend peripherally into the sensory organ-the muscle spindles. T h e capillaries of the muscle spindle are of special interest because they are identical in size and character to capillaries in peripheral nerves," and because their arterioles a n d venules of origin provide a more direct circulation than d o those of extrafusal m ~ s c l e .These '~ capillaries might thus provide a model for the study of some features of the blood -nervous system barrier. T h e present study compares the permeability of intrafusal (IF) capillaries and intramuscular endoneurial capillaries with that of extrafusal (EF)
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capillaries of skeletal muscle using the tracer protein horseradish peroxidase (HRP). Also assesed is the permeability to HRP of the perineurium-like capsule of the spindle. Rabbit spindles from tenuissimus muscle were chosen for study because they consistently contain one or more capillaries inside the periaxial space.2 MATERIALS AND METHODS
Forty New Zealand white rabbits weighing from 1 to 3 lbs (2.2-6.6 kg) received HRP injections or served as controls. HRP (Type 11, RZ 1.5) was obtained from Sigma Laboratories, St. L.ouis, MO. Six rabbits were initially tested for hypersensitivity to HKP. After inhalation anesthesia with methoxyflurane (Penthrane, Abbott Laboratories, North Chicago, IL), the fur on the abdomen was clipped short, arid 6 in1 of Evans blue dye was injected intravenously. Immediately following this, 5, 50, and 100 mg of HRP in 0.1-ml aliquots of saline were in-jected intradermally on the abdomen for comparison with similar 0.1-ml injections of saline alone and of saline containing 10 or 100 mg of histamine. Approximately 30 min following the intradermal injections, the maximum diameter of the blue wheal was measured.
Sensitivity Testing.
Capillary Permeability to HRP. T h e rabbits
that were
to receive intravascular HRP were anesthetized as
described above; the fur was then removed from the abdomen and from both hind extremities. Next, both tenuissimus muscles were exposed over most of their length. The otherwise-undisturbed muscles were then covered again by adjacent muscle and skin. A heating lamp and a warming pad were used to help maintain body temperature. T h e abdominal aorta was then exposed and injected with either 100 or 400 mg of HKP per kilogram body weight in 1 ml of isotonic saline. At predetermined intervals between 1 and 40 min postinjection, a tenuissimus muscle was removed and pinned at a slightly stretched length in 5% glutaraldehyde buffered by 0.1 M sodium cacodylate, pH 7 . 3 , for 2 hr at room temperature. After washing in the same buffer, strips of EF muscle overlying muscle spindles were carefully microdissected free until the spindle capsule was exposed. T h e entire muscle spindle, with the underlying undisturbed bed of EF muscle and the adjacent nerve, was then removed en bloc. The specimens were then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB) to which fresh hydrogen peroxide had been added.'l I n some specimens, the spindle cap-
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Muscle Spindle Capillaries
sule was opened to ensure better exposure of I F capillaries to DAB. T h e 5pecimens were postfixed in 2% osmium tetroxide for 2 hr, after which they were dehydrated by passage through graded ethanol and embedded in Epon. Ultrathin sections were cut on an LKB Ultratome I11 and were examined electron microscopically. Some sections were stained on the grid with saturated uranyl acetate and lead citrate, but most were left unstained so that the distribution of the reaction product could be more accurately defined. Spindles from non-HKP-injected animals were treated in an identical manner: this included incubation in DAB. Capsule Permeability to HRP. I n a few rabbits that had not been injected with HRP, a search was made of the tenuissimus muscle in situ for living muscle spindles; the dissecting microscope was used for this purpose. When a spindle was located, most of the overlying EF fibers were carefully removed. Spindles exposed in this manner retain their tonic and phasic reactivity to muscle stretch, as determined by in-vivo recording from the intramuscular nerve. The spindle and the adjacent EF muscle were then periodically flooded by a solution containing 1 mg of HRP per milliliter to test for permeability of' the spindle capsule to the externally applied tracer. The spindles were removed at varying intervals up to 45 rnin, at which point they were fEed and processed as indicated above. RESULTS
The diameters of Ebans blue stain around the injection sites of saline and 5 mg HRP were equa,l and never exceeded 2.5 mm. In four of the six animals that had been tested for sensitivity, the stain at the 50-mg HKP site was also less than 2.5 mm, while in the other two animals it averaged 5.5 mm. The stain at the 10-mg histamine site averaged 11 mm. It was concluded that the rabbit had minimal sensitivity to Sigma 'l'ype I1 IHRP. We failed t o detect any clinical manifestation of hypersensitivity to the intraaortic injections of HRP, even in quantities of 400 mg/kg. There were no fatalities to anesthesia. Sensitivity to HRP.
Preparation of transverse sections of the processed tissue blocks provided a convenient means of comparing capillaries in the periaxial space of the spindle with those between adjacent EF muscle fibers (fig. 1A). It immediately became evident that the former were larger in diameter and in area. Higher magnification of ultrathin sections (fig. 1B) showed that the typical IF capillary Controls.
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Figure I . (A) Transverse section of a spindle and surrounding extrafusal (EF) muscle, showing two periaxial capillaries (black arrowheads) and EF capillaries (arrows). Other structures of the spindle are the outer capsule (OC), periaxial space (psj, inner capsule (IC), myelinated nerve (N), nuclear bag fiber (nb), nuclear chain fiber (nc), and sensory terminals (St). Phase contrast, bar = 25 pm. (B) Transverse section of an intrafusal capillary in the periaxial space illustrates overlapping region of encircling fibrocytic processes (arrowhead), longitudinal coliagen bundles (col), multilayered basement membrane (bm), enclosing endothelial cells and pericytic processes (pc). and part of a red blood corpuscle (rbc). Treated with tannic acid en bloc; stained with lead citrate and uranyi acetate on the grid. Bar = I pm.
was ensheathed by thin fibrocyte-like processes which were joined by tight junctions where two processes overlapped. Small bundles of longitudinal collagen fibers were situated under the sheath. Collagen also encircled the capillary. 'I'he basement membranes of the endothelial cells were often multilayered. T h e membranes usually divided to surround the many pericytic processes that heavily invested the endothelial cells. Vesicles within the endothelial cells were small and few.*' Following intraaortic injection of HRP, all vessels in the outer portion of the muscle block were filled with the dark reaction product. Central vessels were not demonstrated to be uniformly filled because the DAB failed to penetrate fully into the central core of the larger specimens. EF vessels were in varying stages of patency
Capillary Permeability.
Muscle Spindle Capillaries
or collapse. In the specimens that had been placed in fixative 60 and 90 sec postinjection, the reaction product was entirely contained within the capillary. .4t that time, adluminal pinocytotic vesicles were fdlcd (fig. 2). ,4t slightly later intervals, the cytoplasmic and then the abluminal vesicles filled. T h e reaction product first appeared outside the capillary in some of thc specimcns that had been placed in fixative 2 rnin after the start of the HRP injection. I n specimens removed at 3 min, some of the reaction product was always found in the extraccllular space (fig. 3A). We did not attempt to establish more precisely the time required for HRP t o cscape from the EF capillary, because the fixation time could not be determined. Simionescu et alZ3determined fixation time for rat cremasteric musclc, using this value as an estimate for fixation time of mouse diaphragm. When the cremasteric
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greater dimension than those in EF muscle (fig. LA). 111 some IF capillaries, portions of the cytoplasm of the cndothelial cells were darkly stained (fig. 3Rj. I n the preparations whcre the capsule had been opened to allow lor better penetration of the DAB into the spindle, the appearance did not differ from that described above fbr the intact spindle. In these experiments, the HRP solution was flooded onto t h e living spindle lying in situ in the E F bed. HRP reactioii- product was found to have penetrated between individual EF muscle iibers arid into the EF capillaries. 'The appearance was similar to that of the capillary studied after intraarterial injection. T h e intracellular clefts and the pinocytotic vesicles, particularly those on the abluniirial surface, were filled (fig. 5A). H RP reaction product surrounded the spindle capsule but did not penetrate into the periaxial region, even after 45 rnin of exposure. In most instances, HKP reaction product w a s found in t h e space between the outer two or three layers of capsule cells (fig. 58). 'l'hese spaces were often wider than the more internal iionpenetrated perineurial cell layers. Only a small proportion of the pinocytotic wsicles of capsule cells were filled with reaction product. H RP reaction product was found adjacent to the I F muscle fibers at the distal pole of only one spindle. 'I'his specimen had an obvious mechanical disruption of the outer capsule layer. Intrafusally, thc HRP coricentration was higher under the area of disruption but was absent in the region opposite the disruption. Additional observations of capsule permeability to HRP were made from spindles removed from rabbits that had been injected intraaortically with HRP. In these animals, the HKP that escaped from EF capillaries migrated to the spindle and bathed the capsule for periods of up to 45 min. T h e results were identical to those obtained from spindles flooded with HRP as recorded above: HRP was found between the outer two to three capsule cell layers but never within the periaxial space.
Capsule Permeability.
Figure 2 Extrafusal (EF) capillary at 1 min postinjection HRP reaction product ( r p ) IS in adluminal vesicles and in luminal extent of the interceliular deft but not in extracellular space Unstained, bar = 7 pm
preparation was covered with a parafor-maldehyde-glutaraldehyde fixative, the circulation of red blood corpuscles ceased in 25 sec. Fixation of other preparations may require longer intervals. In other studies (W.R. Kennedy, unpublished data) we have noted, while recording from the sensory nerve of a fully exposed muscle spindle, that the action potentials comprising the sensory response to stretch continued for approximately 2.5 min following direct flooding with a 5% glutaraldehyde solution. It was not our intention in these experimciits to establish the timing or route of escape of' HRP from EF vessels. HRP reaction product was also consistently present in the capillaries of the muscle spindles and in the endoneurial capillaries of the central nerve in the tenuissimus muscle. The reaction product appeared flocculent and was obviously of lower concentration than that found in the EF capillaries (figs. 3B and 4). Reaction product never appeared within the periaxial space outside IF capillaries; nor did it appear outside the endoneurial capillaries, even when the interval following injection was 40 min, although reaction product could be demonstrated within the lumina of these vessels at all times. All capillaries within the spindle and within the nerve were widely patent. The vesicles in the capillaries were only rarely found to contain HRP reaction product. As determined previously, capillaries from the spindle and nerves were of
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Muscle Spindle Capillaries
DISCUSSION
T h e permeability of blood vessels in muscle spindles has not been previously investigated. Only recently was the microcirculation to spindles found to be separate from that t o the surrounding EF muscle.13Artenoles to spindles are third- to f-ourthorder branches from the central muscle artery, whereas most arterioles to EF capillaries are sixth-
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Figure 3 [A) Extrafusai (EF) capillary 3 min postinjection HRP reaction product (rp) has leaked out into surrounding fissue Unstained, bar = 7 p.m (6') intrafusai capiliary 3 rnin postinjection HRP reaction product (rp) remains within the iumen, none has leaked into the periaxial space (psi Unstained, bar = 7 pn
t o eighth-order branches. T h e final arteriolc ends
in two or three capillaries going only to the spindle. These capillaries d o not anastomose wit.h capillaries in EF muscle. I n addition, spindle capillaries were discovered to be structurally different from muscle capillaries." Spindle capillaries are larger than EF capillaries because of a greater endothelial cell complement. T h e clefts between the endothelial cells are consistently found t o have tight junctions, arid the vesicles within thc cells are fewer. Pericyte and basement rnenibraric coverage is heavier. Most of these features are shared with capillaries of peripheral nerve and brain. In addition, the spindle, like nerve, has a perineurialcell-like barrier. separating it from the surrounding extracellular space." These similarities raise the expectation that the permeability 01' IF vessels might be similar to that of vessels in the endoneurium and in the central nervous system. For example, certain dyes and proteins that escape frorn epirieurial blood vesscls remain inside endoneurial vessels of'the rat and mouse.I6 There is species variation, however, for these substances leak out of endo-
Muscle Spindle Capillaries
neurial capillaries of the chicken and guinea pig.'6325 Vessels of the dorsal root ganglion are permeable in all specime~is."'~~ Olssonl' also found some leakage of Evans blue conjugated t o albumin from endoneurial capillaries of the rabbit. I t is well known that horseradish peroxidase and lanthanum are prevented from leaking out of capillaries in -the brain and peripheral nerves by tight junctions between adjacent endothelial cell^.^,'^*^^ T h e results of the present experiments extend this observation to capillaries within t.he muscle spindle, and extend the findings of Olssori and KeeseI8 to include endoneurial capillaries of intramuscular nerve. The floccular nature of the reaction product in IF and endoneurial capillaries suggests that these vessels contain a lower concentration of HRP than do EF capillaries. This difference was just as evident when 100 mgikg was injected as it was with the 400-mgikg infusion. It can be logically argued that HRP might leak out of IF and endoneurial capillaries if the concentrat.ion were further increased. At the high concentration of 400 mg/kg
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Figure 4 Endoneurial capillary 3 min postinjection HRP reaction product (rp) remains within the lumen Unstained, bar = 1 pni
used in these studies, one would expect some leakage or a more prominent presence of HRP in endothelial cell vesicles or intercellular clefts if permeability were possible. The absence of such h i d ings is evidence of'a difference in permeability that serves as a functional barrier for IF capillaries. The finding of tight junctions between adjacent endothelial cells of IF capillaries would appear to corroborate our conclusions regarding the presence of a tight barrier to HRP. The blood pressure and blood flow ineasurements of Fronek and Zweifach,x performed in EF blood vessels of varying size and distance from the main arteriole in cat tenuissirnus muscle, have led us to suspect that blood flow and pressure are high in spindle capillaries as compared to EY capillaries. The large I F capillaries arise directly from thirdorder arterioles, whereas the smaller EF capillaries arise from sixth- to eighth-order arterioles.'3 The apparently lower concentration of HRP in these vessels, suggested by the floccular nature of the reaction product, would be compatible 5vit.h high blood How. Studies of vessels in the inner ear following in-
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Muscle Spindle Capillaries
travasciilar infusion of HRP provide an analogy to our observations regarding IF capillaries. HKP does not leak out of vessels of the basilar membrane, the spiral ligament, 01- the spiral prominence.6~26 In these capillaries, HRP reaction product appeared to be of low concentration and had the same fiocculilr appearance seen in spindle capillaries.6 In the vessels of the adjacent stria vascularis, reaction product filled the lumen as a dense accumulation, also crossing the endothelial cell walls by vesicular transport to extracellular space.6The areas bathed by the capillaries in the basilar membrane, the spiral ligament, and the spiral prominence contain nerve tissue. By inspection, these vessels have the same morphologic f-eatures described herein for spindle and nerve capillaries. In this respect, the observations that blood flow in the spiral ligament was faster than that in the vessels of the stria vascularis of the cochlea20 may be relevant to blood flow in the spindle. Periaxial capillaries are commonly found in humans as well as in cats (personal observations), rabbits,2 and chickens,1s arid are very occasionally found in rats.1° I n these species, capillaries that are presumed to be entering the spindle often run initially for varying distances between layers of capsule cells. Capillaries between the innermost layers in rat and rabbit spindles retain HRP, but those between outer layers leak IIRP (personal observations). Spindles of some smaller species-e.g., rats,'O mice,7 hamsters (personal observation), and snakesg-are nourished by capillaries between layers of capsule cells or by capillaries completely outside the spindle. In the latter spindles, it is presumed that the capsule cells regulate passage of materials from the outside vascular supply to the I F muscle and nerves. Therefore, it would not be too surprising if capsule permeability were found to vary from species to species. The peririeurial cells have generally been accepted as forming a diffusion barrier between extracellular space and eiidoneurium or periaxial space." Electron-microscopic studies have shown that the perineurium prevents penetration of Kerritin4 and €IRP" into the endoneurium. This was confirmed in the present study, where it was dcmonstrated that the inner layers of peririeurial cells of the spindle capsule are a barrier to I I R P . Tracer did enter between the outer two or three cell layers. Olsson and ReeseIxmade the same observation for nerve perineuriuni, but Waggener et alz4found that ferritin did 1101pass even the outer cell layer. It was suggested that the smaller HRP molecule could have passed between incomplete outer layers
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Figure 5 (A) Muscle soaked in HRP for 45 min HRP (arrowhead) has entered the extrafusal (EF) capillary from extracellular space Red blood corpuscle (rbc), reaction (rp) also indicated Unstained. bar = I pni (5) Spmdle soaked i n HRP for 45 rnin HRP is between the outer few capsule layers but dld not penetrate fnto the penaxfal space lntrafusal muscle fiber (‘lF-m), intrafusal capillary (if-cj, outer capsule (OC), and reaction product (rp) are also indicated Lead otrate stained, bar = 7 ~ . c m
of capsule cells.’* We found the outer layer continiious in the few observations made of transverse sect.ions of the entire spindle capsule diameter. It is possible that HRP entered between the outer capsular layers after leaking out of capillaries between the same layers. Neither Olsson and Keese” nor we could est.imate the extent to which HRP underwent vesicular transpor-t across cell layers, but it may be limited, since only a few vesicles contained peroxidatic activity. Another possible pathway by which HRP could penetrate the outer layers of the spindle and nerve perineurial cell layers is t.hrough the clefts of adjacent cells. This would occur if the clefts became more “tight,” progressing from outer to inner capsule layers. A comparison by the freeze-fracture methods of tight junctions in different cell layers would help clarify this possibility, for a relationship does seein to exist between the number of linear strands per zonula occludens and the leakiness of various epithe1ia.j Although Akert et all have demonstrated that the perineurial sealing is of an in-
Muscle Spindle Capillaries
termediate type between “very leaky” and “very tight,” a gradation between cell layers was not mentioned. In summary, t h e permeability characteristics of the capillaries and the capsule of the muscle spindle, as indicated by the use of the tracer protein IIRP, are very similar to those of peripheral nerve. ‘I‘his indicates to us that the normal function of’intrafusal muscle fibers, as well as their sensory arid motor innervation, requires a special intrafusal environment that probably closely resembles the endoneurial environment of the peripheral nerve, these two spaces being in direct continuity. W’e suggest that additional information about the bloodnervous tissue barrier can be obtained by study of the circulation of muscle spindles.
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2. Banks RW, James N T : T h e blood supply of' rabbit muscle spind1es.j Anal 114:7-12, 1973. 3. Brightman MW, Reese TS: Junctions between intimately apposed cell membranes in the vertebrate brain.] Ce/l Biol 4O:648 -675, 1969. 4. Burke1 CV: The histological fine structure of perineurium. A I C ~Rec L ~ 158:177-189, 1967. 5. Claude P, C;rodenongh DA: Fr-acture faces ot 7onulae occhdentes fkom "tight" and "leaky" epithelia. J Cell Bzol 58:390-400, 1973. 6. D u ~ a l lAJ, Quick CA, Sunderland CR: Horseradish peroxidase in the lateral cochleai- ~ v a l l . A n hOto/~uyigol93:304 316, 1971. 7. Edrvai-ds KY: An ultra~tructur-alstudy of neiiromi~scular spindles in normal mice.,/ Anmt 120:149- 168, 1975. 8. Fronek K , Zweifach BW: Microvascular blood flow in cat tcnuissimus muscle. ~ M i c r o z ~ eR . ~ c~ 12:181 A -189, 1977. 9. Fukami Y. Hunt CC: Structure of snake muscle spindles. J ,Vrurophy+d 33:9-27, 1970. 10. James N T , .Meek GA: T h e blood supply d r a t muscle spindlcs.,/Anat 110:164-165. 1971. 11. Karnovsky MJ: T h e ultrastructure basis of capillaq pel-meability studied with peroxidase as a tracer.J Cell B i d 35213 236, 1967. 12. hliyoshi T, Kennedy WR: Morphometric comparison of capillaries in muscle spindle, nerve. and muscle. Arch N e w roc, i n press. 13. Miyoshi T, Krnnedy WK: Microvasculature of rabbit muscle spindles. Arch Seurol, in press. 14. @I$endorf W H : Permeability of the blood-brain barrier. I n Tower DB (Editor): The ,\'~~~oousSy~tem,Vol 1. New York, Raven Press, 1975, pp 279-289. 15. Olsson Y : Studies on vascular permeability in peripheral nerves. I. Distribution of circulating fluorescent serum albumin i n normal, crushed, sectioned peripheral nerve. Arta
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Nrurtrpnthol (Berlin) 7 : l -15, 1 W i . 16. Olsson Y:Phylogenetic variations in the vascular pernleability of peripheral i i e r ~ e st o serum albumin. ,.lctn Pathol .Microhid Scund 69:621 -623, 1967. 17. Olsson Y:Studies on vascular permeability in peripheral rienes. .4cta lVeuropatho/ (Berlin) L5:l 14-126, 1971. 18. Olsson Y , Rcese '13: Permeability of vasa nervoriim and perineurium in mniisr sciatic nen'e studied hy fluorescence and electron microscopy. ,/ Nrwopntlzo/ E x p Nr71rd 30: I05 119, 1971. spindle cap19. Ovalle \.t'K: Fine striicture of the avia~~-muscle sule. Cell Tivsue Rrs 166:285-298. 1976.
20. Perliman HG, Kumura KS: Observations of the living blood vesels 01. the cochlea. Ann Otol Rhino/ Laqvzgol 64: 1176 1192, 1955. 21. Reese TS, Karnovsky MJ: I h e structural localiLation o f a b h d - b r d i I l harrier tn exogenous peroxidase. J Cell Z3zol 34:244-266, 1967.
22. Shanta TR, Bourne GH: T h e perineurial epithelium-a new concept. I n Bourne GH (Editor): T h f Sfntcturr crnd Funclion uf h e N e w o u c T i ~ s u r ,Vol 1. N e w York, Academic Press. 1968, pp 279-459. 23. Siriiionescu M. Sirriionescu N, Palade CE: Morphometric data on the endotheliurn of blood capillaries. J Cell B i d 60:128-132, 1974.
24. Waggerier JD, Bunri SH, Beggs J: T h e diffusion of t'erritin within the peripheral nelve sheath. An clectron microscopy stiidy.,] Nruropnthol k:xp Nvirrol 24:430-443, 1965. 25, Waksman HH: Experimental study of diphthcric polyneuritis in the rabbit and guinea pig. 111. T h e blood-nerve barrier in the 1abbit.j iVeuroputho1 Exp Neicrol 20:35-77, 1961. 26. Winther PP: l ' h e permeability of the guinea pig cochlear capillaries to horseradish peroxidase. Z %e/!/orsch 114:193 202, 1971.
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