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Basal lamina at the site of spinal cord injury in normal, immunotolerant and immunosuppressed rats
Neuroscience Letters, 54 (1985) 225-230 Elsevier Scientific Publishers Ireland Ltd.
225
NSL 03165
BASAL LAMINA AT THE SITE OF SPINAL CORD INJURY IN NORMAL, IMMUNOTOLERANT AND IMMUNOSUPPRESSED RATS
E.R. FERJNOA'·2.*, T.P. KOWALSKI 2 and H.L. VAHLSINO,,2
'Neurology Service oj the Veterans Administration Medical Center and Department of Neurosciences, University oj California, San Diego, CA and 2Departments oj Pathology and Neurology, Veterans Administration and University oj Michigan Medical Centers, Ann Arbor, MI (U.S.A.) (Received August 3rd, 1984; Revised version received and accepted November 29th, 1984)
Key words: basal lamina - cyclophosphamide - regeneration - spinal cord - peroxidase-antiperoxidase stain - immunologically unresponsive - rat The cut ends of a rat spinal cord are capped with basal lamina (BL) within 20 days. This BL may block regenerating axons. BL at the transection site in rats made immunologically unresponsive to central nervous system antigens is not significantly different from that of control rats, but rats treated with cyclophosphamide show a less complete BL cap during the first 25 days. This may account for the increased axonal regeneration found in cyclophosphamide-treated rats.
While it is generally agreed that axons within the central nervous system (eNS) have the capacity to regenerate, functionally significant regeneration of long ascending or descending axons in the mammalian spinal cord does not occur. Many hypotheses have been offered to explain this phenomenon. We have suggested that the basal lamina (BL) formed at the lesion site may act as a barrier to regenerating axons [3]. In untreated rats, BL caps the transected end of the cord 20 days after transection. Growing axons may penetrate BL only with difficulty [8, 12]. Usually the BL deflects or impedes the progress of growing axon tips. Since a cap of BL forms before axons of the cord are likely to regrow, BL may be an impediment to regeneration. Some treatments allow a minimal amount of long tract regeneration after spinal cord transection in the rat [1, 2, 4-7]. Significant regeneration was clearly demonstrable but not sufficient to permit functional recovery. How did these treatments facilitate limited regrowth of long tract axons? Did the treatments affect BL formation at the injury? This experiment reports light microscopic observations of an immunohistochemically stained BL and electron microscopic (EM) observations of BL in the area of injury. Treated animals are compared to matched controls. "Author for reprint requests at present address: Chief, Neurology Service (127), Veterans Administration Medical Center, 950 15th Street, Augusta, GA 30910, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
226
Rats and operative procedures have been described previously [3]. Treatment groups. Three groups of rats were studied. A control group of 15 rats received no special treatment. A group of 15 rats was treated with a single 75 mg/kg dose of cyclophosphamide (CY) 48 h after spinal cord transection. This treatment allows some long tract regeneration [1, 5]. A third group of 15 rats was made immunologically unresponsive to CNS antigen as previously described. Rats treated this way also showed some regeneration [4]. Three animals from each group were studied at 5, 10, 15, 25 and 30 days after spinal cord transection. Tissue preparation and BL staining technique. On the date of sacrifice, animals were heparinized, anesthetized and perfused with fixative [3]. The cord was removed and placed in fixative at 4-6°C for 4 h, then transferred into 5070 phosphatebuffered sucrose, pH 7.4, overnight. Spinal cord containing the area of transection was cut in two longitudinally. One half was used for frozen sections studied by light microscopy; 15 /lm longitudinal frozen sections were transferred to gelatin-coated slides, air dried and stained using the previously described highly specific rat epithelial BL staining techniques [9]. Six sections from each animal were stained. The quality of staining could be assayed by the stain on adjacent normal pia-glial BL, an internal positive control. The other half of the cord was tissue-chopped into 150 /-Lm thick longitudinal slabs. Adjacent tissue chopped pieces were processed in one of two ways for EM study. (1) A tissue-chopped piece was dehydrated, embedded in Araldite and semithin 1 /lm sections stained with methylene blue in 1070 sodium borate were used to locate the interface between relatively healthy CNS tissue and scar tissue. The area of interface was identified and, if fixation was adequate, thin sections were cut, stained with uranyl acetate and lead citrate, and studied with a Zeiss EM 10 electron microscope. (2) One adjacent tissue-chopped section from an animal in each treatment group at each time interval was stained en bloc with the immunohistochemical stain for BL to provide a direct test of the specificity of the stain at the ultramicroscopic level. Evaluation procedure. We blindly evaluated 6 longitudinal sections from each animal in groups of 3 animals together, always including one animal from each treatment group. Attempts to measure directly the percent of the cut surface covered with newly formed BL were frustrated by the irregular nature of the border between viable CNS tissue and the scar. Finger-like projections of preserved nervous tissue and small and large cysts abounded at the border. On section, the border is represented by multiple circular profiles as well as the more regular cut surface boundary. Therefore, the observer estimated (to the nearest 5070) the amount of surface area on which BL could be identified: An average percent for each animal was determined before the blind was broken. Occasionally, failure of the stain (as evidenced by failure to stain the normal pia-glial BL - an internal control) or poor quality of the available frozen sections made evaluation of one or two slides on an animal impossible. On only one animal (from the 5-day control group) was the
227
number of well-stained slides too few to determine a reliable estimate of the percent BL capping. This animal was dropped from analysis. All animals were evaluated in an identical manner and therefore any errors intrinsic in this subjective method should not affect our ability to detect differences between groups. Available, I-pm sections of plastic-embedded cord were searched to locate areas of interface between viable CNS and scar. We then noted the presence or absence of BL in adjacent ultrathin sections. We also searched the injury area for BL not associated with the pia-glial surface or the glial interface with surviving blood vessels. In the thin sections from those tissue-chopped pieces which had been stained en bloc, we checked for specificity of the BL stain and the presence of stain on newly formed BL in the area of injured spinal cord interface with scar. The results of light microscopic evaluation of immunohistochemically stained sections (Fig. 1) are listed in Table I. Utilizing Student's r-test, we compared each treatment group to controls. The immunotolerant group was not different from controls. The group treated with CY, however, was less completely capped with BL than the control group (P= 0.04).
Scar
Viable eNS
Fig. I. Basal lamina capping of the cut end of the spinal cord at 15 days in an animal treated with cyclophosphamide. Segments of BL, indicated by arrows, cap parts of the cut surface. On survey of the entire cut surface, about 15-20070 was covered with BL. PAP indirect antibody stain technique, dark-field illumination. Because the sections evaluated were relatively thick (15 /Lm) and high power was necessary to visualize the reaction product of the stain, the photographic image is not in sharp focus at all depths. Microscopic magnification x 400. Total magnification (microscopic plus photographic) x 1600.
228 TABLE I PERCENT OF CUT SURFACE OF THE TRANSECTED SPINAL CORD WHICH WAS CAPPED WITH BL AT SPECIFIED TIMES AFTER SURGERY Immunotolerant rats did not differ from the control group. Cyclophosphamide-treated rats have less BL cap when compared to controls (P= 0.04). Control (0= 14)
5 days
average 10 days
average IS days
average
25 days
average
35 days
average
Immunotoleraot (0 = IS)
Cyclophosphamide (n= 15)
8 8
19 14 2
8 7
8
11.7
6.7
5
15 12 7
21 12 12
17 12 8
11.3
15
12.7
41 30 20
34 30 27
25 22 16
30.3
30.3
21
69 61 50
55 48 30
47 30 22
60
44.3
33
70 66 25
75 50 47
55 52 45
53.7
57.3
50.7
Evaluation with EM was more difficult. Fixative frequently did not reach the injured area quickly enough to provide ideal fixation. Fixation was so poor at 5 days that satisfactory EM could not be done. Eight of 9 animals studied at 10 days could be evaluated. No injury area BL was found. By IS, 25 and 35 days, some BL was seen in 6 of 9 animals. At the magnifications required to visualize BL, it was impossible to quantitate the percent of interface with non-eNS tissue which had a BL. However, little or no injury area BL was seen at 10 days post-surgery; more, at 15 days; at 25 and 30 days, injury area BL was identified in most animals. EM sections stained en bloc provided assurance that the peroxidase-antiperoxidase (PAP) stain was attached to BL of the pia-glial surface and injury area BL (Fig. 2). We previously found that BL completely capped the cut spinal cord 20 days after spinal cord transection [3]. In this experiment, the BL cap was still incomplete in
229
Cyst of scar
Fig. 2. Basal lamina, indicated by arrows, covers reactive glial cells at the border between CNS and nonCNS scar tissue. AS, astrocyte; DM, degenerating myelin. The BL is stained with the PAP indirect antibody stain technique. Note the specificity of the stain and the close relation of the injury area BL to the glial surface. Original magnification x 4000. Magnification of print x 7330.
all animals at 35 days. This reflects a change in the pia-glial BL specific antisera used. Because the hyperimmune serum used in this experiment was different than that used previously, we cannot directly compare the two experiments. Treatment with a single injection of 75 mg/kg of CY significantly slows the development of the BL cap over the end of the cut spinal cord. It is possible that this treatment delays the capping of the cord long enough to allow the earliest few axons to regenerate. Later, a more complete BL cap may block additional axons which attempt to regenerate. This hypothesis allows for a minimal amount of regeneration and explains why more complete regeneration was not found. The mechanism by which CY disturbs the production of BL is unclear. CY primarily affects rapidly dividing cells but also affects the growth and metabolism of most, if not all, cells. A delay in the formation of BL at the site of transection was therefore expected. This experiment does not prove that inhibition of formation of BL is the mechanism by which Cy' treatment facilitates axonal regeneration, but it does suggest this may be a part of the mechanism. Matthews et al. [10, 11] have reported no long-range effect of CY on the EM appearance of the collagenous scar or the investing glial limiting membrane which demarcates the scar from viable CNS tissue in treated spinal cord transected rats. However, at 45 days, they did note a transient reduction in the fibrous connective tissue of the scar in treated rats. Their rats received two 15 mg/kg doses of CY, one dose on the day of surgery and another 24 h later. We have not tried this dose schedule, but we have used chronic lower dose CY (1.5 mg/kg daily and 5 mg/kg daily) and failed to find regeneration [2, 7]. Matthews et al. [11] did note a two-fold increase in the number ofaxons infiltrating the scar matrix 90 days after transection in animals treated with CY. This may be due to a direct enhancement of axonal growth instead of, or along with, a decrease in the presumed mechanical barriers which block axons. They did not report any changes in BL. We cannot determine
230
if this is because of the limited field they could study by EM, or if their findings reflect the dose-treatment schedule differences. This study illustrates that the BL formed at the injury site after spinal cord transection as demonstrated by our immunohistochemical stain is delayed in appearance in those animals treated with a single high dose of CY 48 h after spinal cord transection. No similar delay of the appareance of BL is seen in animals previously made immunologically unresponsive to eNS antigens. The authors wish to express their appreciation to Ms. Linda Lee Austin and Ms. Luann Woodward for technical and photographic assistance. They also wish to acknowledge the secretarial assistance of Mrs. Diane Trakas, Ms. Barbara Reader, Ms. Marlene Brindell and Mrs. Emily Maheu. This project was supported by the Veterans Administration Research Service and by the Development Funds of the University of Michigan Medical Center Pathology Department, Ann Arbor, ML I Feringa, E.R., Davis, S.W., Vahlsing, H.L. and Shuer, L.M., Fink-Heimer/Nauta demonstration of regenerating axons in the rat spinal cord, Arch. Neurol., 35 (1978) 522-526. 2 Feringa, E.R., Johnson, R.D. and Wendt, J.D., Spinal cord regeneration in rats aftger immunosuppressive treatment, Arch. Neurol., 32 (1975) 676-683. 3 Feringa, E.R., Kowalski, T.F. and Vahlsing, H.L., Basal lamina formation at the site of spinal cord transection, Ann. Neurol., 42 (1980) 148-154. 4 Feringa, E.R., Nelson, K.R., Vahlsing, H.L. and Dauser, R.C., Spinal cord regeneration in rats made immunologically unresponsive to CNS antigens, J. Neurol. Neurosurg. Psychiatr., 42 (1979) 642-648. 5 Feringa, E.R., Shuer, L.M., Vahlsing, H.L. and Davis, S.W., Regeneration of corticospinal axons in the rat, Ann. Neurol., 2 (1977) 315-321. 6 Feringa, E.R., Vahlsing, H.L. and Dauser, R.C., Orthograde flow of tritiated proline in corticospinal neurons at various ages and after spinal cord injury, J. Neurol. Neurosurg. Psychiatr., 47 (1984) 917-920. 7 Feringa, E.R., Wendt, J.S. and Johnson, R.D., Immunosuppressive treatment to enhance spinal cord regeneration in rats, Neurology, 24 (1974) 287-293. 8 Kao, C.C., Chang, L.W. and Bloodworth, J.M.B., Axonal regeneration across transected mammalian spinal cords: an electron microscopic study of delayed microsurgical nerve grafting, Exp. Neurol., 54 (1977) 591-615. 9 Kowalski, T.F., Vahlsing, H.L. and Feringa, E.R., Light microscopic, immunohistochemical localization of the pia-glial basal lamina, 1. Histochem. Cytochem., 28 (1980) 347-353. 10 Matthews, M.A., St. Onge, M.F., Faciane, c.L. and Gelderd, J .B., Axon sprouting into segments of rat spinal cord adjacent to the site of a previous transection, Neuropathol. Appl. Neurobiol., 5 (1979) 181-196. 11 Matthews, M.A., St. Onge, M.F., Faciane, C.L. and Gelderd, J.B., Spinal cord transection: a quantitative analysis of elements of the connective tissue matrix formed within the site of lesion following administration of piromen, cytoxan or trypsin, Neuropathol. Appl. Neurobiol., 5 (1979) 161-180. 12 Stensaas, L.J. and Feringa, E.R., Axon regeneration across the site of injury in the optic nerve of the next Triturus pyrrhogaster, Cell Tiss. Res., 179 (1977) 501-506.
225
NSL 03165
BASAL LAMINA AT THE SITE OF SPINAL CORD INJURY IN NORMAL, IMMUNOTOLERANT AND IMMUNOSUPPRESSED RATS
E.R. FERJNOA'·2.*, T.P. KOWALSKI 2 and H.L. VAHLSINO,,2
'Neurology Service oj the Veterans Administration Medical Center and Department of Neurosciences, University oj California, San Diego, CA and 2Departments oj Pathology and Neurology, Veterans Administration and University oj Michigan Medical Centers, Ann Arbor, MI (U.S.A.) (Received August 3rd, 1984; Revised version received and accepted November 29th, 1984)
Key words: basal lamina - cyclophosphamide - regeneration - spinal cord - peroxidase-antiperoxidase stain - immunologically unresponsive - rat The cut ends of a rat spinal cord are capped with basal lamina (BL) within 20 days. This BL may block regenerating axons. BL at the transection site in rats made immunologically unresponsive to central nervous system antigens is not significantly different from that of control rats, but rats treated with cyclophosphamide show a less complete BL cap during the first 25 days. This may account for the increased axonal regeneration found in cyclophosphamide-treated rats.
While it is generally agreed that axons within the central nervous system (eNS) have the capacity to regenerate, functionally significant regeneration of long ascending or descending axons in the mammalian spinal cord does not occur. Many hypotheses have been offered to explain this phenomenon. We have suggested that the basal lamina (BL) formed at the lesion site may act as a barrier to regenerating axons [3]. In untreated rats, BL caps the transected end of the cord 20 days after transection. Growing axons may penetrate BL only with difficulty [8, 12]. Usually the BL deflects or impedes the progress of growing axon tips. Since a cap of BL forms before axons of the cord are likely to regrow, BL may be an impediment to regeneration. Some treatments allow a minimal amount of long tract regeneration after spinal cord transection in the rat [1, 2, 4-7]. Significant regeneration was clearly demonstrable but not sufficient to permit functional recovery. How did these treatments facilitate limited regrowth of long tract axons? Did the treatments affect BL formation at the injury? This experiment reports light microscopic observations of an immunohistochemically stained BL and electron microscopic (EM) observations of BL in the area of injury. Treated animals are compared to matched controls. "Author for reprint requests at present address: Chief, Neurology Service (127), Veterans Administration Medical Center, 950 15th Street, Augusta, GA 30910, U.S.A. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
226
Rats and operative procedures have been described previously [3]. Treatment groups. Three groups of rats were studied. A control group of 15 rats received no special treatment. A group of 15 rats was treated with a single 75 mg/kg dose of cyclophosphamide (CY) 48 h after spinal cord transection. This treatment allows some long tract regeneration [1, 5]. A third group of 15 rats was made immunologically unresponsive to CNS antigen as previously described. Rats treated this way also showed some regeneration [4]. Three animals from each group were studied at 5, 10, 15, 25 and 30 days after spinal cord transection. Tissue preparation and BL staining technique. On the date of sacrifice, animals were heparinized, anesthetized and perfused with fixative [3]. The cord was removed and placed in fixative at 4-6°C for 4 h, then transferred into 5070 phosphatebuffered sucrose, pH 7.4, overnight. Spinal cord containing the area of transection was cut in two longitudinally. One half was used for frozen sections studied by light microscopy; 15 /lm longitudinal frozen sections were transferred to gelatin-coated slides, air dried and stained using the previously described highly specific rat epithelial BL staining techniques [9]. Six sections from each animal were stained. The quality of staining could be assayed by the stain on adjacent normal pia-glial BL, an internal positive control. The other half of the cord was tissue-chopped into 150 /-Lm thick longitudinal slabs. Adjacent tissue chopped pieces were processed in one of two ways for EM study. (1) A tissue-chopped piece was dehydrated, embedded in Araldite and semithin 1 /lm sections stained with methylene blue in 1070 sodium borate were used to locate the interface between relatively healthy CNS tissue and scar tissue. The area of interface was identified and, if fixation was adequate, thin sections were cut, stained with uranyl acetate and lead citrate, and studied with a Zeiss EM 10 electron microscope. (2) One adjacent tissue-chopped section from an animal in each treatment group at each time interval was stained en bloc with the immunohistochemical stain for BL to provide a direct test of the specificity of the stain at the ultramicroscopic level. Evaluation procedure. We blindly evaluated 6 longitudinal sections from each animal in groups of 3 animals together, always including one animal from each treatment group. Attempts to measure directly the percent of the cut surface covered with newly formed BL were frustrated by the irregular nature of the border between viable CNS tissue and the scar. Finger-like projections of preserved nervous tissue and small and large cysts abounded at the border. On section, the border is represented by multiple circular profiles as well as the more regular cut surface boundary. Therefore, the observer estimated (to the nearest 5070) the amount of surface area on which BL could be identified: An average percent for each animal was determined before the blind was broken. Occasionally, failure of the stain (as evidenced by failure to stain the normal pia-glial BL - an internal control) or poor quality of the available frozen sections made evaluation of one or two slides on an animal impossible. On only one animal (from the 5-day control group) was the
227
number of well-stained slides too few to determine a reliable estimate of the percent BL capping. This animal was dropped from analysis. All animals were evaluated in an identical manner and therefore any errors intrinsic in this subjective method should not affect our ability to detect differences between groups. Available, I-pm sections of plastic-embedded cord were searched to locate areas of interface between viable CNS and scar. We then noted the presence or absence of BL in adjacent ultrathin sections. We also searched the injury area for BL not associated with the pia-glial surface or the glial interface with surviving blood vessels. In the thin sections from those tissue-chopped pieces which had been stained en bloc, we checked for specificity of the BL stain and the presence of stain on newly formed BL in the area of injured spinal cord interface with scar. The results of light microscopic evaluation of immunohistochemically stained sections (Fig. 1) are listed in Table I. Utilizing Student's r-test, we compared each treatment group to controls. The immunotolerant group was not different from controls. The group treated with CY, however, was less completely capped with BL than the control group (P= 0.04).
Scar
Viable eNS
Fig. I. Basal lamina capping of the cut end of the spinal cord at 15 days in an animal treated with cyclophosphamide. Segments of BL, indicated by arrows, cap parts of the cut surface. On survey of the entire cut surface, about 15-20070 was covered with BL. PAP indirect antibody stain technique, dark-field illumination. Because the sections evaluated were relatively thick (15 /Lm) and high power was necessary to visualize the reaction product of the stain, the photographic image is not in sharp focus at all depths. Microscopic magnification x 400. Total magnification (microscopic plus photographic) x 1600.
228 TABLE I PERCENT OF CUT SURFACE OF THE TRANSECTED SPINAL CORD WHICH WAS CAPPED WITH BL AT SPECIFIED TIMES AFTER SURGERY Immunotolerant rats did not differ from the control group. Cyclophosphamide-treated rats have less BL cap when compared to controls (P= 0.04). Control (0= 14)
5 days
average 10 days
average IS days
average
25 days
average
35 days
average
Immunotoleraot (0 = IS)
Cyclophosphamide (n= 15)
8 8
19 14 2
8 7
8
11.7
6.7
5
15 12 7
21 12 12
17 12 8
11.3
15
12.7
41 30 20
34 30 27
25 22 16
30.3
30.3
21
69 61 50
55 48 30
47 30 22
60
44.3
33
70 66 25
75 50 47
55 52 45
53.7
57.3
50.7
Evaluation with EM was more difficult. Fixative frequently did not reach the injured area quickly enough to provide ideal fixation. Fixation was so poor at 5 days that satisfactory EM could not be done. Eight of 9 animals studied at 10 days could be evaluated. No injury area BL was found. By IS, 25 and 35 days, some BL was seen in 6 of 9 animals. At the magnifications required to visualize BL, it was impossible to quantitate the percent of interface with non-eNS tissue which had a BL. However, little or no injury area BL was seen at 10 days post-surgery; more, at 15 days; at 25 and 30 days, injury area BL was identified in most animals. EM sections stained en bloc provided assurance that the peroxidase-antiperoxidase (PAP) stain was attached to BL of the pia-glial surface and injury area BL (Fig. 2). We previously found that BL completely capped the cut spinal cord 20 days after spinal cord transection [3]. In this experiment, the BL cap was still incomplete in
229
Cyst of scar
Fig. 2. Basal lamina, indicated by arrows, covers reactive glial cells at the border between CNS and nonCNS scar tissue. AS, astrocyte; DM, degenerating myelin. The BL is stained with the PAP indirect antibody stain technique. Note the specificity of the stain and the close relation of the injury area BL to the glial surface. Original magnification x 4000. Magnification of print x 7330.
all animals at 35 days. This reflects a change in the pia-glial BL specific antisera used. Because the hyperimmune serum used in this experiment was different than that used previously, we cannot directly compare the two experiments. Treatment with a single injection of 75 mg/kg of CY significantly slows the development of the BL cap over the end of the cut spinal cord. It is possible that this treatment delays the capping of the cord long enough to allow the earliest few axons to regenerate. Later, a more complete BL cap may block additional axons which attempt to regenerate. This hypothesis allows for a minimal amount of regeneration and explains why more complete regeneration was not found. The mechanism by which CY disturbs the production of BL is unclear. CY primarily affects rapidly dividing cells but also affects the growth and metabolism of most, if not all, cells. A delay in the formation of BL at the site of transection was therefore expected. This experiment does not prove that inhibition of formation of BL is the mechanism by which Cy' treatment facilitates axonal regeneration, but it does suggest this may be a part of the mechanism. Matthews et al. [10, 11] have reported no long-range effect of CY on the EM appearance of the collagenous scar or the investing glial limiting membrane which demarcates the scar from viable CNS tissue in treated spinal cord transected rats. However, at 45 days, they did note a transient reduction in the fibrous connective tissue of the scar in treated rats. Their rats received two 15 mg/kg doses of CY, one dose on the day of surgery and another 24 h later. We have not tried this dose schedule, but we have used chronic lower dose CY (1.5 mg/kg daily and 5 mg/kg daily) and failed to find regeneration [2, 7]. Matthews et al. [11] did note a two-fold increase in the number ofaxons infiltrating the scar matrix 90 days after transection in animals treated with CY. This may be due to a direct enhancement of axonal growth instead of, or along with, a decrease in the presumed mechanical barriers which block axons. They did not report any changes in BL. We cannot determine
230
if this is because of the limited field they could study by EM, or if their findings reflect the dose-treatment schedule differences. This study illustrates that the BL formed at the injury site after spinal cord transection as demonstrated by our immunohistochemical stain is delayed in appearance in those animals treated with a single high dose of CY 48 h after spinal cord transection. No similar delay of the appareance of BL is seen in animals previously made immunologically unresponsive to eNS antigens. The authors wish to express their appreciation to Ms. Linda Lee Austin and Ms. Luann Woodward for technical and photographic assistance. They also wish to acknowledge the secretarial assistance of Mrs. Diane Trakas, Ms. Barbara Reader, Ms. Marlene Brindell and Mrs. Emily Maheu. This project was supported by the Veterans Administration Research Service and by the Development Funds of the University of Michigan Medical Center Pathology Department, Ann Arbor, ML I Feringa, E.R., Davis, S.W., Vahlsing, H.L. and Shuer, L.M., Fink-Heimer/Nauta demonstration of regenerating axons in the rat spinal cord, Arch. Neurol., 35 (1978) 522-526. 2 Feringa, E.R., Johnson, R.D. and Wendt, J.D., Spinal cord regeneration in rats aftger immunosuppressive treatment, Arch. Neurol., 32 (1975) 676-683. 3 Feringa, E.R., Kowalski, T.F. and Vahlsing, H.L., Basal lamina formation at the site of spinal cord transection, Ann. Neurol., 42 (1980) 148-154. 4 Feringa, E.R., Nelson, K.R., Vahlsing, H.L. and Dauser, R.C., Spinal cord regeneration in rats made immunologically unresponsive to CNS antigens, J. Neurol. Neurosurg. Psychiatr., 42 (1979) 642-648. 5 Feringa, E.R., Shuer, L.M., Vahlsing, H.L. and Davis, S.W., Regeneration of corticospinal axons in the rat, Ann. Neurol., 2 (1977) 315-321. 6 Feringa, E.R., Vahlsing, H.L. and Dauser, R.C., Orthograde flow of tritiated proline in corticospinal neurons at various ages and after spinal cord injury, J. Neurol. Neurosurg. Psychiatr., 47 (1984) 917-920. 7 Feringa, E.R., Wendt, J.S. and Johnson, R.D., Immunosuppressive treatment to enhance spinal cord regeneration in rats, Neurology, 24 (1974) 287-293. 8 Kao, C.C., Chang, L.W. and Bloodworth, J.M.B., Axonal regeneration across transected mammalian spinal cords: an electron microscopic study of delayed microsurgical nerve grafting, Exp. Neurol., 54 (1977) 591-615. 9 Kowalski, T.F., Vahlsing, H.L. and Feringa, E.R., Light microscopic, immunohistochemical localization of the pia-glial basal lamina, 1. Histochem. Cytochem., 28 (1980) 347-353. 10 Matthews, M.A., St. Onge, M.F., Faciane, c.L. and Gelderd, J .B., Axon sprouting into segments of rat spinal cord adjacent to the site of a previous transection, Neuropathol. Appl. Neurobiol., 5 (1979) 181-196. 11 Matthews, M.A., St. Onge, M.F., Faciane, C.L. and Gelderd, J.B., Spinal cord transection: a quantitative analysis of elements of the connective tissue matrix formed within the site of lesion following administration of piromen, cytoxan or trypsin, Neuropathol. Appl. Neurobiol., 5 (1979) 161-180. 12 Stensaas, L.J. and Feringa, E.R., Axon regeneration across the site of injury in the optic nerve of the next Triturus pyrrhogaster, Cell Tiss. Res., 179 (1977) 501-506.