Biomedical subjects
Publications and source records attributed to J J Bernstein.
A patient developed signs of a progressive pericardial effusion over a period of eight days. Diagnostic and therapeutic pericardiocentesis was performed. Viral titres to psittacosis rose from less than 1:8 to greater than 1:256 indicating recent infection.
IgG-positive astrocytes have been reported in scrapie-induced and Alzheimer's cortical plaques, multiple sclerosis, and CNS tissue around abscesses, metastatic tumors and primary tumors of glial origin. The present experiments ascertain if this immunoglobulin positivity is specific for these cases or a function of astrocytes around any site of injury in the CNS. The spinal cords of 30, 300-g Sprague-Dawley male rats were lesioned by passing a 26 gauge needle through the cord at T6. After periods as long as 9 months, the spinal cords were processed for paraffin immunohistochemistry with antisera to IgM, IgG or double labeled for these immunoglobulins and GFAP (glial fibrillary acidic protein) a specific cytoplasmic marker for astrocytes. From 1 through 7 days after lesioning double labeled astrocytes in and around the site of injury are both IgM- and IgG-positive. From 14 days through 9 months postlesion, double labeled astrocytes surrounding the lesion are only positive for IgG. These data indicate a relationship between immunoglobulin availability, continued blood-brain barrier perturbation to immunoglobulins and the ability of reactive astrocytes to sequestor immunoglobulins. IgM is an early determinant for reactive astrocytes and IgG positivity is determinant for reactive astrocytes at any time period.
The present experiment examines astrocytes in fetal cerebral cortical homografts to adult rat spinal cords. The purpose of this study is to determine if astrocytes are structurally organized within the graft. Also, the presence or absence of astrogliosis may be an indicator of the metabolic status of the graft. Embryonic cerebral cortex was taken at 14 days gestational age and transplanted into adult spinal cord at the level of the sixth thoracic vertebra. The homografts were examined at the light and electron microscopic levels from 7 days postimplantation (PI) to 6 months PI with glial fibrillary acidic protein antiserum which is a specific immunohistochemical marker for astrocytes. At 7 days PI, immunoreactive astrocytes were present only at the periphery of the graft and appeared to be associated with blood vessels. By 30 days PI, normal protoplasmic astrocytes were present throughout the graft. No hypertrophied astrocytes are present at 30 days PI, but the numerical density of astrocytes is greater than that of normal cerebral cortical gray matter. Fibrous astrocytes are present in the periphery of the implant and many of these astrocytes extended their processes between the host and the graft. Occasional glial scarring is observed between the gray matter of the host and graft, but generally no glial scar occurred in the interface between the graft and the host gray matter. By 45 days PI, hypertrophied astrocytes can be seen in the graft, but are confined in this age group to perivascular regions.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of ischemia (removal of oxygen and glucose for 4 h) and anoxia (removal of oxygen alone) on astrocytes were studied in dissociated cultures of E14 spinal cord containing both neurons and astrocytes. In addition, a group of cultures was treated with a low Na+, low Ca2+, and high K+ medium during the 4-h ischemic period (ischemia-protected group), a process that protects neurons from ischemic damage under identical conditions. Astrocytes were examined immunohistochemically using glial fibrillary acidic protein (GFAI) antiserum 24 h after insult. Densitometry and statistical analysis (1-way analysis of variance [ANOVA], a priori; 2-tailed Tukey-t, a posteriori) of the digitized images of the somata and processes of astrocytes in the anti-GFAP reacted cultures showed significant differences between the groups; a significant increase (P less than 0.01) in the GFAP-positive reaction in the somata of ischemic astrocytes and a significant decrease (P less than 0.01) in the GFAP-positive reaction in the processes of ischemic, ischemia-protected, and anoxic astrocytes. There were no significant differences in the GFAP immunoreactivity of somata between control, ischemia-protected, and anoxic astrocytes or of processes from ischemic, ischemia-protected, and anoxic astrocytes. These data show that following ischemia cultured astrocytes increase somatic GFAP immunoreactivity compared to all other groups tested whereas the staining intensity for GFAP was decreased in the processes of all three experimental groups compared to controls. Ischemia protection resulted in the absence of the enhancement of somatic GFAP immunoreactivity. The relationship of the astrocytic response and the type of cellular stress is discussed.
Fourteen-day gestation fetal cerebral cortex homografts were transplanted into the thoracic (T6) spinal cord between the left dorsal column and dorsal horn of adult host rats. The transplants were soaked in 2.0 micrograms/ml of the lectin Phaseolus vulgaris leucoagglutinin (PHAL) prior to implantation. Transplanted host spinal cords were utilized at 7, 14, and 24 d and at 1 and 2 months postimplantation. Paraffin-sectioned spinal cords were double labeled for PHAL and glial fibrillary acidic protein (GFAP) by using FITC- and RITC-conjugated secondary antisera, respectively. Montages of FITC- and RITC-positive cells were analyzed for cells containing both fluorescences. Double-labeled cells (PHAL-GFAP) were transplant-derived astrocytes. Transplant-derived astrocytes were observed to initiate migration in the white matter columns of the host at approximately 14 d after transplantation. Double-labeled astrocytes were observed in cervical and lumbar spinal cord of the host (ca. 3.5 cm away from the center of the transplant) at 2 months postoperative. These astrocytes migrated at approximately 0.76 mm a day (after a 14-d delay). At 2 months, transplant-derived astrocytes composed as much as 50% of the astrocytes in the white matter of the host 2.0 mm from the transplant. The migrated astrocytes were hypertrophied and appeared reactive. Astrocytes in spinal gray matter only migrate about 1.0 mm from the graft-host interface. Transplant-derived astrocytes can migrate the entire length of the spinal cord white matter.
Fetal spinal cord grafts survive for long periods in host spinal cord. We explored the ability of such grafts into a lesion in C3 fasciculus gracilis to influence the expected deterioration of hind limb performance following this lesion. Ten adult Sprague-Dawley rats were placed on a schedule of 23 h off and 1 h on water. These subjects were then trained to traverse a narrow platform for a water reward. Criterion was established as 10 trials/day completed in 15 s a trial for 2 consecutive days. Animals were ranked for hind limb and forelimb performance utilizing slips, recovery, and manner of traversing the platform. In a triple-blind experimental design, the animals' identification was coded and laminectomy performed at C3. The fasciculus gracilis was bilaterally transected at the rostral and caudal borders of the segment and aspirated. Five subjects were chosen at random immediately after the lesion was made for the implantation of two 1-mm segments of E14 spinal cord from fetuses of timed pregnant Sprague-Dawley dams at the time of the lesion. Coded lesion and lesion-transplant animals were then tested after 21, 30, 45, 60, and 90 days until criterion or for 4 days. Hind limb performance of the lesion-only (ANOVA chi 2 = 13.04, df 5; P less than 0.05) and lesion-transplant groups (ANOVA chi 2 = 11.82, df 5; P less than 0.05) showed that the C3 fetal transplants significantly decreased (P less than 0.05) the severity of hind limb deficit at 21 days postlesion and significantly (P less than 0.05) decreased the expected foot slips and limb recovery to the platform that was expected and did develop in lesion-only animals at 90 days. There were no significant differences in forelimb performance (ANOVAs P greater than 0.05). There were few small-caliber nerve fibers in the fasciculus gracilis at the level of C1 indicating that reinnervation was not a major factor in the reduction of the deficit.
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Cell suspensions from 14-day gestation rat spinal cord can be successfully cultured on collagen gels containing laminin. After 7 and 14 days in culture the gels were removed from the dish and slices of gel with cultured cells were transplanted into the dorsal column of adult rats. Thirty days later there was no evidence of the gel. However, grafted neurons with well-developed organelles, axons, dendrites, axosomatic and axodendritic synapses were observed in the white matter of the host dorsal columns. Oligodendrocytes and astrocytes were also observed in the graft. This histocompatible substrate for culturing fetal CNS is a mechanism for transplanting cultured CNS cells as grafts of known maturity and synaptic organization into adult host CNS.
Fetal central nervous system homografts to adult spinal cord are considered a potential aid for recovery of function after paraplegia. This study utilizes somatostatin (SOM) immunohistochemistry to study the organization of an embryonic day 14 (E14) neocortical homograft into the spinal cord of an adult host over 6 postoperative months. Although the E14 homograft does not contain SOM-positive cells, SOM-reactive neurons are expressed by 30 days postimplantation and are still present in 6-month-old homografts. SOM-immunoreactive neurons are bitufted or multipolar and have dendrites that are confined to the graft. The homograft contains SOM-immunoreactive axons entering and/or exiting from lamina II in the host dorsal horn and SOM-positive homografted neurons send axons into the host ventral columns. These data show that the SOM peptide neocortical phenotype is preserved in homografts to spinal cord but there is anatomical host-homograft integration.
Transplants of central nervous system to adult spinal cords are considered as potential aids in regeneration of the spinal cord and/or recovery of function after injury. The organization and development of the implant are important issues in seeking the potential for a transplant and host to become functionally integrated. This study uses embryonic cerebral cortex transplanted into the spinal cord of adult rats (T6) and examined the development and organization of the transplant with an antibody to vasoactive intestinal polypeptide (VIP). The cell bodies of VIP neurons are in the implants at 30 days postimplantation, but few of the somata have processes. By 45 days postimplantation, VIP neurons in the implant have dendrites and axons and are clearly recognizable as cortical bipolar cells which are not normally present in the thoracic spinal cord. These data show that neurons in embryonic cerebral cortical implants into the spinal cord elaborate the appropriate biochemical and morphological constituents in spite of the ectopic location. However, the cell processes develop at a slower than normal pace. Morphological interaction between the host spinal cord and the implant can be demonstrated possibly as early as 45 days postimplantation and clearly at 6 months following the implant. Thus, further examination of cerebral cortical implants as a potential aid in alleviation of paraplegia subsequent to spinal cord injury is warranted.
Xenografts of embryonic day 5 chick cortex were placed into the dorsal column of the spinal cord of 15 adult male Sprague-Dawley rats. This implantation site is known to support comparable E11 rat cortex for months. At 1 DPI (day postoperative), transplanted chicken cortex formed neuroepithelia with a surround of transplanted cells. By 3 DPI, all transplanted cells contained cytoplasmic lipid droplets or lysosomes, as well as necrotic cells. By 7 DPI, the fetal chicken cortical xenograft had necrosed or was phagocytized. The rapid cross-class xenograft rejection is unique since grafts of various rodent tissues tend to survive in the adult rodent central nervous system.
The ependymal cells in rat host spinal cord were studied after transplantation of fetal cerebral cortex homografts. Adult Sprague-Dawley male rats had pieces of E14 fetal rat cortex pressure-injected into the spinal cord at T6. The tissue was prepared for light and electron microscopy and studied for over 2 months postimplantation. At 7 and 14 days postimplantation (DPI), there were dividing ventricular-ependymal cells lining cysts in the host spinal cord. After 21 days, cell division was no longer observed in these cells, and only mature ependymal cells lined these cysts. Mature ependymal cells were either: columnar, pseudocolumnar, cuboidal, squamous, or pseudomorphic; had cilia and villi on only one surface of the cell and shared tight junctions when contiguous. These data show that ependymal cells were mature cellular components of adult spinal cord after transplantation of fetal CNS grafts. In addition, ventricular-ependymal cells continued to divide within the parenchyma of the graft, indicating continued growth of transplanted fetal cerebral cortex in host spinal cord.
Allografts of mature rat superior cervical ganglia (SCG) survived for up to 120 days following transplantation to regenerating and degenerating peripheral nerves of adult rats. Transplants contained the constituents of normal superior cervical ganglia and there was evidence of outgrowth of catecholamine containing fibers from the transplants. However, fibers did not attain the length necessary to enter peripheral muscles and no muscle response could be elicited by electrical stimulation of the implanted nerve where the transplanted mature SCG was the only source of nerve fibers.
Fetal CNS (cortex and spinal cord) transplants survive and grow in the spinal cord of adult rats. The present experiments study the initial stages (10 days) to determine the developmental pattern of early (E11) fetal cortex and spinal cord transplants implanted into the T6 spinal cord of adult rats (n = 50). Controls were age-matched fetuses or pups from the donor females and surgical and vehicle controls (n = 20). E11 fetal cortex (0.5 X 1.0 mm pieces of neuroepithelium) or spinal cord (1.0 mm) were dissected from caesarean delivered fetuses from time pregnant Sprague-Dawley rats. The fetal tissue was pressure-injected between the dorsal horn and dorsal column of the host. Hosts were prepared for light and electron microscopy 1, 3, 7 and 10 days postimplantation (DPI). At 1 DPI, ovoid, ventriculated multiple neuroepithelia formed in an implantation-induced cyst in the host spinal cord. The inner layer of cells lining the ventricle of the ovoid neuroepithelia was actively dividing. The nuclei of the cells differentiated as they reached the surface of the neuroepithelium. This pattern was repeated at 3-10 DPI. At 7-10 DPI, differentiated neurons and neuroglia surrounded actively dividing neuroepithelia. These cells comprised the bulk of the transplant in the cyst. Transplanted mesenchymal cells formed pockets of cartilage in the implanted spinal cord. Although transplanted CNS consisted of only neuroepithelial cells, they differentiated into neurons that were typical for cortex or spinal cord. These data indicate that the transplanted E11 fetal tissue formed neuroepithelia which established the pattern of growth for these transplants.
Basal lamina is reconstructed over the lesioned surface of the spinal cord. The following experiment (90 rats) studies the ultrastructure of the formation of this membrane and the immunohistochemistry of laminin production (a major secreted component of basal lamina). After hemisection of the spinal cord at T6 animals were prepared for electron microscopy or antilaminin-biotin-avidin-peroxidase incubation. Three-5 days posthemisection, antilaminin reaction product was observed in astrocytes and their processes which faced the lesion, endothelia of blood vessels or pia. Ultrastructurally (3 days), basal lamina was polymerizing as small projections on the surface of astrocytic membranes facing the lesion, endothelia or pia. By 5 days the basal lamina was a single membrane, folded multiple sheets or in swirls. At 6-10 days the antilaminin reaction and the basal lamina (except for duplications) did not differ from normal. Reactive astrocytes secrete laminin for at least 3-5 days after hemisection and form basal lamina on the lesioned surface of the spinal cord after spinal cord hemisection.
The effect of spinal dorsal funicular lesions (T 12) upon the frequency of boutons on, and diameters of the somata of pyramidal cells in layer VB of hindlimb cortex was studied. Adult rats sustained bilateral damage to either the dorsal column (DC, n = 10) alone or DC combined with the corticospinal tract (CS) (DC + CS, n = 34) and were utilized 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperatively (DPO). Neurons randomly sampled from 44 lesioned and 13 unoperated cases were analyzed for the number of silver-impregnated boutons (Rasmussen method) on the circumference of the soma as well as diameters of the soma, nucleus, and nucleolus. Analyses of variance comparing across lesioned and normal groups were significant for bouton counts on the soma (P less than 0.01), and diameters (long axis) of somata (P less than 0.01) and their nuclei (P less than 0.05). Both lesioned groups exhibited significant decreases from normal for these latter three parameters. With respect to survival time for the DC + CS-lesioned animals we noted the following: (1) Bouton counts on the soma significantly decreased below normal between 1 and 60 DPO; this decrease was most dramatic during the first three days postlesion. (2) Somal diameter (long axis) significantly decreased below normal between 2 and 120 DPO (except at 14 and 90 DPO). (3) Nuclear diameter (long axis) significantly decreased below normal only at 90 DPO. (4) Bouton counts on somata of neurons in layers VB and IV [Ganchrow and Bernstein, 1981] of hindlimb cortex correlated negatively and significantly across 120 postlesion days. The rapid shrinkage and reduced afferentation of layer VB somata during the first week following DC + CS lesions suggest initial, retrograde reactions to CS axotomy. Since bouton counts on layer VB somata were significantly less (P less than 0.05) in DC- than DC + CS-lesioned rats, it is hypothesized that CS axotomy regulated a set-point for increased afferentation which was maintained on the shrunken somata between 7 and 120 DPO.
Morphological aspects of the implantation of 11-day-old fetal (E11) rat cortex into regenerating peripheral nerves of adult rats were analyzed during the first 3 weeks after transplantation. On day 11 of gestation, donor fetuses were removed from one uterine horn of pregnant Sprague-Dawley rats and pieces of cortex (1.0 X 0.5 mm) were dissected from them for transplantation. Fetuses and normally delivered pups from the other uterine horn were used as age-matched controls. In 45 adult male Sprague-Dawley rat hosts (200 to 300 g), the epineurium of the nerve to the biceps femoris muscle was crushed and incised, the perineurium minced, and E11 donor cortex was injected into the nerve. At 1 to 7 days postimplantation (DPI), undifferentiated neuroepithelial cells were dispersed or in oval neuroepithelial-like structures throughout the regenerating nerve. At 7 to 14 DPI, neurons and neuroglia were differentiating and interspersed among fascicles of regenerating host axons. At 14 to 21 DPI, aggregates of differentiated neurons surrounded by neuropil containing a rich synaptic bed were interspersed with regenerated host peripheral axons. Fetal cortical implants survived, increased in size, and differentiated throughout the postoperative period, matching their timed controls.
The growth, differentiation and function of E11 fetal cortex was studied over 4 months after implantation into the degenerating nerve to the biceps femoris muscle of 70 rats. At 7 days postimplantation (DPI) the implant formed a neuroepithelium which contained mainly undifferentiated cells within a reconstituted perineurium. At 14-21 DPI the neurons contained differentiated, mature nuclei, poorly developed Nissl bodies and normal cytoplasmic organelles. Neuroglia were also observed with typical nuclei and a dense cytoplasmic ground. At 30 days the implants reached their greatest degree of differentiation. Neurons had mature nuclei and cytoplasmic organelles. There were few axosomatic synapses or neuronal cell processes. Neuroglia were prominent with many cell processes. The neuropil contained myelinated and unmyelinated nerve fibers, dendrites and many axodendritic synapses. At 40-50 DPI the neurons and neuronal cell processes were degenerating. At 60-120 DPI the implant contained only neuroglia. Stimulation of only the implant resulted in biceps femoris muscle contraction in 5 animals at 30 DPI and one animal at 40, 45 and 50 days. Injection of 10 microliter of WGA-HRP into the biceps femoris muscle resulted in at least 20 neurons in 3 30-day implants that contained HRP reaction product. These data show that fetal cortical neurons can assume functions different from normal, functionally reinnervate adult muscle and can differentiate and form an ectopic nervous system for at least 30-days after implantation into a degenerating adult host peripheral nerve.