Biomedical subjects
Publications and source records attributed to H L Vahlsing.
One year after a T9 spinal cord transection, horseradish peroxidase was inserted into the spinal cord at T3-T4. Only about 7% of the number of corticospinal neurons labeled in control rats were labeled in exactly matched transected rats. This long-term loss of labeled neurons makes cell death the most likely explanation for the failure to identify corticospinal neurons in spinal-cord-transected rats.
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.
Cell death of embryonic neurons which are unable to attain a proper target is well established. A delayed cell death of adult neurons permanently separated from their target tissue has been demonstrated for several cell groups. Cells of the dorsal root ganglia (DRG) are unique in that a single T-shape neurite has a peripheral branch which extends (for root L5) to the hind limb and a central branch extending into the spinal cord. We found a significant loss of L5 DRG neurons 25 weeks after hind limb amputation. This finding is consistent with the hypothesis that neuron survival is dependent on connection with a suitable target. We were unable to detect cell death in the DRG of L5 after complete spinal cord transection at T9. Separation of DRG cells from their central target is unimportant to neuronal survival.
The death of embryonic central nervous system (CNS) neurons deprived of a target is well established. In adult rats, similar cell death of corticospinal and rubrospinal motor neurons occurs as a delayed response to spinal cord transection. We document the loss of neurons in Clarke's column, secondary ascending spinocerebellar neurons in adult rats, after complete spinal cord transection at T-9. Twenty-five weeks after spinal cord transection, horseradish peroxidase (HRP) studies showed a dramatic loss of labeled cells in rats with transected spinal cords as compared to matched control rats. Cresyl echt violet-stained sections failed to support the hypothesis that unlabeled cells persist in a shrunken, inactive state; instead we found far fewer identifiable neurons in Clarke's column. Although we saw little gliosis in the area of cell loss, gliosis was evident in the adjacent corticospinal tract which was severed in the original surgical injury. Amputation of the right hind limb resulted in a paradoxical increase in labeled Clarke's column cells on the right. Total cells stained with cresyl echt violet in amputated animals were not different from right to left. The increase in labeled cells on the amputated side may have been caused by an increase in metabolic activity of these deafferentated neurons which resulted in more effective axoplasmic transport of the HRP label.
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This electron microscopic study confirms that basal lamina (BL) begins to cap the cut end of the spinal cord 15 days after spinal cord transection. BL is first seen immediately adjacent to reactive glial cells but only when there is collagen in the nearby interstitial space. This finding suggests that collagen may provide a trigger to initiate the production of BL by reactive glia. We found no direct evidence that BL in this injury area impeded the outgrowth of regenerating neurites.
The amount of radioactive proline which reaches the cervical cord by axoplasmic flow after intracortical injection of label is higher in rapidly growing 3 to 6 week old rats but becomes relatively constant in unoperated control rats beyond age 10 weeks. In adult rats with spinal cord transection at T-8, however, the amount of tritiated proline detected in the cervical cord above the site of transection is markedly increased five weeks after surgery, falls to more normal levels by 14 weeks after surgery, and is significantly below normal at 25 weeks after surgery. These findings are consistent with abortive attempts to regenerate axons at five weeks after injury. Twenty-five weeks after injury neuronal death and loss of both cells and axons which would normally project to the caudal cord through the site of spinal cord transection result in a decrease in the axon label found in the cervical region. Recognition of this variability in the amount of radioactivity that reaches the cervical region after spinal cord injury forced a reconsideration of previously reported evidence for regeneration in spinal cord transected animals receiving no specific postoperative therapy. There is no evidence for regeneration in such untreated transected rats.
Dying cortical neurons, identified by standard histologic criteria, were observed in the rat sensory/motor cortex after spinal cord transection. The peak incidence of these changes was 10 weeks after injury. At that time, spinal-cord-injured animals showed 10 times as many abnormal cells as were found in matched controls (p less than or equal to 0.05). Dying cells were found in the same anatomic location as corticospinal neurons axotomized by the experimental injury, and at the expected time after injury. The survival of corticospinal neurons in these young adult rats may be dependent on obtaining a crucial input from an appropriate target cell (neuron).
Complete spinal cord transection at T-6/T-7 in rats caused a decrease in the number of surviving corticospinal neurons. Cell death began 5 and 10 weeks after cord injury. The number of surviving cells decreased progressively for at least 25 weeks after injury. Surviving cells were identified by their ability to transport horseradish peroxidase (HRP) retrograde from a T-1/T-2 insertion site to cortical cell somas. Therapy aimed at promoting corticospinal tract regeneration must be started early after spinal cord injury.
The rates of axoplasmic transport were studied in the corticospinal tract of the rat by injecting tritiated proline into the sensory-motor cortex and subsequently analyzing the distribution of incorporated label in the spinal cord at intervals after injection. A mathematical model of the anatomy of the corticospinal tract was developed and used in analysis of the data. The rate of a fast component was calculated to be 240-420 mm per day, which is comparable with rates of fast components in the peripheral nervous system (PNS), but considerably greater than rates in other tracts in the central nervous system. A slow component was calculated to have a transport rate of 3-8 mm per day which is greater than rates found either in the CNS or PNS. This higher rate may be related to the greater length of the corticospinal tract as compared to other CNS tracts studied.
The pia-glial basal lamina (BL) at the site of spinal cord injury could be an important physical impediment to central nervous system regeneration. We used an epithelial BL-specific immunohistochemical stain to determine the location of the pia-glial BL after spinal cord transection. Small segments of BL were found at the margin of the lesion 5 days after transection. After 10 days, longer and more numerous segments were seen. At 20 days, the entire transected end of the spinal cord was capped by a layer of BL.
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The current histologic methods for studying the pia-glial basal lamina (BL) are inappropriate for high contrast, permanent light microscopy preparation. We have developed a staining technique for epithelial BL which is highly specific, extremely sensitive, permanent, relatively inexpensive, and suitable for light or electron microscopy (EM). Central nervous system (CNS) basement membrane zone (BMZ) antigens were isolated by the technique of Meezan (1975) from female albino Wistar rats. Using this CNS BMZ preparation as an antigenic source, a hyperimmune rabbit serum was developed. This serum was exhaustively adsorbed with rat splenic pulp to remove undesirable antibodies to endothelial BL and collagen. The peroxidase-antiperoxidase indirect antibody technique was used to test the staining specificity of this splenic adsorbed serum on different tissues containing BLs of known origin and/or function. The results indicated that this BL staining technique was specific for epithelial BL of the rat and of some other species.
Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols of Matinian and Andreasian's two most successful treatment regimens, we repeated their experiments using United States produced pure hyaluronidase and trypsin or trypsin and elastase. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiated proline by regenerated corticospinal axons. None of the treated animals differed from control animals treated only with the enzyme vehicle. No animals walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.
Russian investigators have recently reported clinical recovery of enzyme treated, spinal cord transected rats. Using the exact protocols outlined by Matinian and Andreasian, we repeated a portion of their experiment using a Lidase preparation manufactured in the USSR. Animals were evaluated for return of bladder function, clinical evidence of hind limb motor function, cortical evoked response after sciatic nerve stimulation, and axonal transport of cortically injected tritiate proline by regenerated corticospinal axons. The only difference between treated and control animals was that the mean total body weight of the treated animals increased more than that of controls (p less than 0.05). No animal walked, had return of voluntary motor activity, showed cortical evoked response, or had evidence for transport of tritiated proline over regenerated corticospinal axons.
Seventy days after complete spinal cord transection, both treated and untreated rats showed evidence that some corticospinal axons had regenerated. Rats made immunologically unresponsive to CNS tissue showed no increase in corticospinal regeneration as measured by orthograde axoplasmic flow of tritated proline or retrograde axoplasmic labelling with horeseradish peroxidase. However, treated rats did demonstrate electrophysiological evidence of regeneration of long ascending sensory pathways. Tolerant animals additionally treated with cyclophosphamide showed corticospinal axonal regeneration by tritated proline transport and electrophysiological techniques and also showed electrophysiological evidence of ascending sensory tract regeneration.
Six months after complete spinal cord transection, regenerated descending motor axons can be found by electrophysiologic testing and can also be demonstrated anatomically using the Fink-Heimer/Nauta technique. Regeneration was found in all animals, treated or control, but statistically significant increased regeneration as measured by the Fink-Heimer/Nauta technique was found in animals treated with a single injection of 75 mg/kg of cyclophosphamide 24 hours after the original spinal cord transection. This result is similar to those of another study using the orthograde axonal transport of tritiated proline as a measure of axonal regeneration. The mode of action of cyclophosphamide may be by its immunosuppressive properties because this drug is most effective when administered 24 hours after cord transection, a time when its effects as an immunosuppressant would be maximal.
A simple artificial respiration mask for rats was constructed from the drip chamber of an intravenous solution administration set. The mask was held in place by means of straps and a hole through which the maximillary incisors protruded. This mask permitted artificial ventilation during drug-induced respiratory paralysis, was easily and inexpensively constructed, and may be used with a variety of positive pressure respirators.