Nonspecific esterase activity in plastic-embedded tissues using Meldola Blue.

A method for the demonstration of nonspecific esterase activity in plastic-embedded tissues using Meldola Blue is described. Although Meldola Blue does not function as an electron carrier in this method, it may account for the short incubation time and excellent localization of the reaction product. Use of Meldola Blue is an advancement in the demonstration of enzyme activity in plastic sections.

Evaluation of blood vessel and neurite growth into a collagen matrix placed within a surgically created gap in rat spinal cord.

A complete gap of 3-4 mm was surgically created in the spinal cord of adult rats between the T8-T10 vertebral level, filled with a collagen matrix and closed with sutures. Animals were killed at weekly intervals from 14 to 42 days postimplantation. The collagen implant (CI) was evaluated for ingrowth of blood vessels and neurites using light and electron microscopic techniques. At 42 days postimplantation, 3 CI animals underwent a transection at the rostro-caudal center of the CI followed by placement of horseradish peroxidase (HRP)-impregnated filter paper between the cut ends. The number and location of HRP-labeled cells in CI animals were compared to normal animals that received a spinal cord transection at the T9 vertebral level immediately followed by placement of HRP between the cut stumps of spinal cord. Connective tissue (CT) septae, neurites and blood vessels invaded the CI from adjacent spinal neuropil and surrounding CT capsule. CI animals revealed HRP-labeled cells in the intermediolateral cell column (IML), parabrachial nuclei, locus coeruleus and red nucleus with a preponderance of labeled cells found in the IML rostral to the CI. In contrast, no IML were labeled in thoracic spinal cord rostral to HRP implantation in control animals. It was concluded that the above brainstem nuclei, which normally project axons into spinal cord, are capable of extending their severed axons into the CI following spinal cord injury and that uninjured IML cells also sprout axons into the CI.

A preliminary study of homotopic fetal cortical and spinal cotransplants in adult rats.

Neocortical and spinal tissue from a given E16-17 rat fetus were homotopically transplanted into lesion sites of adult rats which had undergone combined cortical and complete lower thoracic spinal cord lesions. Spinal cord transplants were placed either directly into the gap in host spinal cord or embedded in a collagen matrix. Animals were killed from 4 days to 8 months and tissues were processed for light microscopy. All cortical transplants survived and integrated with host brain. Many axons appeared to grow between the cortical transplant and subjacent host parenchyma. Only collagen-embedded spinal transplants survived. At 8 months, two animals underwent spinal cord transection and HRP implantation two vertebral segments rostral to the spinal cord transplantation site. Both animals revealed HRP-labeled neurons in the cortical transplants. It was concluded that 1) homotopically transplanted fetal cortical tissue can survive and may be capable of extending axons to midthoracic levels, and 2) a collagen matrix may enhance the survival of fetal tissues transplanted into a complete gap in host spinal cord.

Effects of exercise on immune functions of undernourished mice.

Regular moderate exercise may modulate the response to a stressor and thus improve immune functions in conditions commonly associated with immunodepression and elevated levels of stress hormones. For example, anorexia nervosa patients, many of whom engage in regular aerobic exercise, generally have normal immune function and viral disease resistance in spite of their severe undernutrition. To test the hypothesis that exercise can prevent undernutrition-induced immunodepression, mice were fed a nutritionally complete, semi-purified diet, either ad libitum or in restricted quantities to induce 25% loss of initial weight over 3 weeks. Half the animals from each dietary group were run on a treadmill for 30 min/day, 5 days/week. Exercise had no effect on several measures of nutritional status. Spleen weight and blastogenic response to lipopolysaccharide were significantly increased by exercise in undernourished mice. In vivo antibody response to sheep red blood cells, and in vitro splenic responses to concanavalin A and phytohemagglutin were not significantly affected by exercise. Serum corticosterone level was increased by food restriction and significantly decreased by exercise in the undernourished mice. Within a treatment group there were no significant correlations between serum corticosterone level and any immune system measure. Hypothalamic concentration of uric acid was increased in food restriction groups and concentration of norepinephrine was increased in exercise groups. The results suggest that regular exercise may help prevent undernutrition-induced immunodepression, possibly through modulation of the stress response.

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: complete decussation apparent.

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye (right eye in Tg419 and left eye in Tt038) had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin.

Quantitative anatomical and behavioral analyses of regeneration and collateral sprouting following spinal cord transection in the nurse shark (ginglymostoma cirratum).

The spinal cord was transected at the mid-thoracic level in 32 nurse sharks. Four animals per group were sacrificed at intervals of 10, 20, 30, 40, 60 and 90 days postoperative. Two groups of fish underwent a subsequent spinla1 cord retransection at the same site at 90 days and were sacrificed 10 and 20 days later. Three sections of spinal cord were removed from each shark for histological analysis. Behaviorally, timed trials for swimming speed and a strength test for axial musculature contraction caudal to the lesion site were performed at 5 day postoperative intervals. Histological analysis showed little regeneration (9-13 percent) of two descending tracts 90 days following the lesion and no return of rostrally controlled movements caudal to the lesion. However, synaptic readjustment did occur caudal to the lesion. This phenomenon was attributed to local segmental sprouting of adjacent, intact nerve fibers. A close correlation was shown between this synaptic readjustment and the strength of uncontrollable undulatory movements seen caudal to the lesion site following spinal cord transection. The relationship of regeneration and collateral sprouting to quantitative behavioral changes is discussed.

Tissue calcium levels following spinal cord transection in rats.

Adult rats were subjected to midthoracic spinal cord transections. Three segments of spinal cord, each approximately 5 mm in length, were removed from each animal at intervals from 5 min to 48 h postlesion; one from the lesion site and one each immediately rostral and caudal to the transection. Total tissue calcium concentrations ([Ca]t) for each spinal cord segment were determined using atomic absorption spectrophotometry and compared to control segments from untransected animals. [Ca]t levels in the segment at the lesion site was significantly elevated above control values at 30 min post-lesion, but decreased to control levels by 1 h. All other segments remained at control levels for the duration of the postlesion period. The rapid rise and fall of [Ca]t at the lesion site differs from spinal cord contusion studies in which [Ca]t remains at elevated levels for extended periods. It is postulated that the "open" transection injury permits the rapid clearance of calcium from the injury site.

Qualitative and quantitative effects of ACTH, piromen, cytoxan and isobutyl-2-cyanoacrylate treatments following spinal cord transection in rats.

Adult, male, Long-Evans hooded rats were subjected to a spinal cord transection in the mid-thoracic region. Following surgery, animals were divided into groups for treatments with ACTH, Piromen, isobutyl-2-cyanoacrylate and Cytoxan. Animals were sacrificed at varying intervals from 7-180 days. Horizontal sections of the lesion site were stained with the Bodian silver and Gomori trichrome techniques and numbers of nerve fibers counted within the lesion. Statistical analysis of the quantitative data indicated that Piromen and Cytoxan, respectively, were significantly more effective than the other treatments in promoting regeneration of nerve fibers into the scar. Microcysts appeared adjacent to the lesion in all animals early in the postoperative period and coalesced to form large cavitations which continued to enlarge throughout the postoperative period. Although nerve fibers appeared to traverse the lesion, no return of function was observed.

The vertebral level of origin of spinal nerves in the rat.

Several dissections were performed to determine the level of spinal cord termination and the vertebral level at which the dorsal and ventral roots of spinal nerves C1-S4 emerged from the spinal cord in the rat. These levels of emergence were then compared to the level of exit from the vertebral canal. The dissections demonstrated that the effect of differential growth between spinal cord and vertebral column begins in the lower cervical region and becomes progressively more pronounced throughout thoracic and lumbar levels. The disparity between the vertebral level of emergence of spinal roots from the spinal cord and their level of exit via intervertebral foramina was found to be considerably larger than was previously reported by Greene ('68). It was further noted that the spinal cord terminated at the level of the intervertebral disc between the third and fourth lumbar vertebrae, not between the fourth and fifth lumbar vertebrae as reported by Greene ('68).

Effect of spinal cord transection on the reproductive system in the female rat.

Cycling rats had their spinal cord transected (SCT) at the T5 vertebral level and/or were unilaterally ovariectomized (ULO) at metestrus, and allowed to cycle either for one estrous cycle or for 19 days. Estrous cycle lengths were increased following SCT in all animals. After one cycle, SCT rats showed no effect on other reproductive parameters studies. In contrast, after 19 days decreases in body and ovarian weights, and number of follicles greater than 448 micrometers and ova shed per ovary were observed. When ULO was performed in conjunction with SCT, compensatory ovarian responses occurred. The data demonstrate that an intact adrenergic system is necessary for normal ovarian function in the intact rat but not necessary for compensatory ovarian responses to occur following ULO.

Spinal cord vasculature of the rat: a histochemical study of the metabolism of arteries and arterioles.

Spinal cord arteries and arterioles of adult female rats were examined histochemically to determine their metabolic profiles. The metabolic pathways evaluated included those related to aerobic (oxidative phosphorylation, Kreb's cycle and respiratory chain) and anaerobic (glycolysis) capacity, hexosemonophosphate-shunt activity, beta-oxidation of fat and adenosine triphosphate utilization. The amounts of deoxyribonucleic and ribonucleic acids were determined as an indication of protein synthesis. The present findings indicate that arteries of the rat spinal cord are metabolically active with high capacities for both aerobic and anaerobic metabolism, and possess a significant potential for nucleic acid and protein synthesis. Lipid catabolism, via beta-oxidation of fat, may serve as one source of energy. The arteries also demonstrate a high capacity for utilization of adenosine triphosphate. In contrast, the spinal cord arterioles show a lower capacity for aerobic metabolism and lipid utilization, while anaerobic glycolysis may be a main source of energy. The arterioles also demonstrate a significant potential for nucleic acid and protein synthesis, in addition to a high capacity for adenosine triphosphate utilization.

Metabolism of coronary vasculature in euthyroid, hyperthyroid and recovering hyperthyroid rats: a histochemical study.

Coronary arteries and arterioles from normal rats, from rats made hyperthyroid by administration of desiccated thyroid for 10 weeks, and from hyperthyroid rats which were then fed normal control diets for 10 weeks, were examined histochemically to determine the activity of key metabolic pathways. The primary aims of this study were to determine if the alterations in particular enzyme and substrate activities that occur in thyrotoxic rat myocardium, arteries and arterioles were reversible and would return to normal levels following cessation of the hyperthyroid state. Our results suggest that hyperthyroid rats, even after 10 weeks on the normal diet, still show some compromise in arteriolar aerobic metabolism in favor of anaerobic pathways, while coronary arteries still demonstrate little glucose-6-phosphate dehydrogenase activity. Myocardial metabolic activity approximates that of normal control animals by the end of the 10th week on the normal diet.

Therapeutic effects of hyperbaric oxygen and dimethyl sulfoxide following spinal cord transections in rats.

Thirty adult, male, Long-Evans hooded rats underwent spinal cord transections at the T5 vertebral level. Following surgery, animals were separated into three groups: Group I received only normal postoperative care; Group II received daily hyperbaric oxygen (HBO) treatments for 47-54 consecutive days; Group III received the same HBO treatment as Group II in addition to subcutaneous injections of dimethyl sulfoxide (DMSO) for 10 consecutive days. All animals were killed 60-70 days postlesion. The lesioned area of spinal cord was removed and prepared for light and electron microscopy. Group I animals showed typical scar reduction of cavitations, increased scarring, and more nerve fibers within the lesion. Three animals in this group exhibited coordinated hindlimb movement, with one animal showing weight-bearing ability. The lesion sit in group III animals revealed a reduction in collagen formation and a further increase in the number of nerve fibers. Six animals in Group III showed coordinated hindlimb movements; among these two displayed weight-bearing ability and sensory return.

Axon sprouting into segments of rat spinal cord adjacent to the site of a previous transection.

Transection of the spinal cord is adult Long-Evans Hooded rats is followed by the formation of a connective tissue matrix in the lesion site and the rapid erosion of the neural elements above and below this zone particularly within the dorsal white columns. In the period between 15--45 days after operation two significant events begin to occur. First the injured surfaces of the divided cord become invested by a glial limiting membrane (glia limitans) and, concomitantly, large numbers of axons ensheathed by Schwann cells sprout into the scar matrix and along the eroded dorsal column region. The injured surface of the spinal cord is highly irregular with deep, collagen-filled rifts into which the sprouting axons may probe and penetrate into the adjacent normal neuropil. Electron microscopic examination generally reveals the interposed glia limitans and that these fibres are usually restricted to the peripheral environment. However, as some axons approach the reconstituting glia limitans, they are enveloped by an astrocytic cytoplasmic process which may either displace the Schwann cell or encompass it together with the enclosed axons. This last phenomenon appears to precede the entry of some axons into the neuropil and suggests that the glia limitans may not necessarily represent an impenetrable barrier to the passage of regenerating axons into the CNS. Apparent maintenance of most of these fibres for periods of up to 3 months may suggest that viable, functional synapses were established upon available neuronal elements, but clear evidence of this could not be documented.

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.

Long-Evans hooded rats were cordotomized at the T-5 level and given either (1) cyclophosphamide (cytoxan), an immunosuppressive, (2) piromen, a bacterial polysaccharide-nucleic acid complex, (3) topical and systemic trypsin, or (4) no further specific treatment. Because of past and present controversy surrounding the proposed ability of these agents to promote spinal cord regeneration, a systematic study, employing light and electron microscopy, and quantitative methods in a single animal model, was done in order to re-evaluate the effects of each treatment upon the connective tissue matrix which forms in the defect left by transection. After an initial inflammatory reaction during the first week after surgery, the lesion zone is characterized either by areas of dense collagenous connective tissue with occasional fibroblasts and macrophages, or a loose areolar tissue with numerous sheets and cords of mesodermal cellular elements but minimal collagen. By 45 days postoperatively (dpo), axons supported by Schwann cells invade and become entangled in the loose connective tissue matrix. With longer postoperative survival, cysts appear craniad and caudad to the lesion and erode much of the scar together with viable neural tissue. Giving cytoxan or piromen did not result in any qualitative alteration of the scar matrix as evidenced by electron microscopy. Quantitative analysis revealed a slight reduction in the fibrous connective tissue component of the scar at 45--90 dpo, but this was transient when longer postoperative periods were studied. Trypsin caused a significant reduction in the amount of fibrous connective tissue with a concomitant increase in loose connective tissue and the appearance of a few distinctive, compact bundles of unmyelinated axons lacking Schwann cells. Consistent behavioral changes were not observed in any group which could distinguish them from the controls. Our results appear to contradict the findings of Matinian and Andreasian (1976) who reported return of normal sensori-motor function in 80% of their animals treated with topical and systemic trypsin. It is concluded that a major impediment to whatever longterm regenerative potential exists within the spinal cord is the lack of axonal guiding elements within the scar, but more importantly, the severe erosion of the remaining spinal cord due to cyst enlargement.

The effects of interleukin-1 receptor antagonist protein (IRAP) infusion following spinal cord transection in rats.

A laminectomy was performed at the T5-T6 vertebral level in adult, male, Sprague-Dawley rats and the spinal cord transected with a scalpel. A group of sham animals was subjected to the same surgery without the transection step. A group of unhandled control rats was also included. A subgroup of transected animals received a subcutaneous osmotic minipump that dispensed IL-1 receptor antagonist protein (IRAP) at the transection site for 7 consecutive days. Another transected subgroup received a minipump that infused the vehicle only. IRAP-treated rats displayed a significant reduction in body temperature (p < 0.05) compared with vehicle-treated rats. The IRAP-treated rats were also less active when assessed for locomotor behavior using an HVS computerized tracking system (p < 0.01). IRAP treatment had no effect on serum corticosterone, beta-endorphin levels, Con A, PHA, or LPS-induced splenocyte mitogenesis when compared with vehicle-treated animals. However, half of the IRAP-treated animals exhibited a substantive reduction in the number of reactive astrocytes near the transection site, suggesting a possible effect of IRAP on astrocyte activation.

A Histochemical study of the coronary vasculature in euthyroid and hyperthyroid rats.

Coronary arteries and arterioles in the left and right ventricles from normal and hyperthyroid rats were examined histochemically to determine and to compare their metabolic activities. The test animals were made hyperthyroid by administration of desiccated thyroid for 8-10 weeks. Using histochemical techniques, selected enzymes and components of key metabolic pathways were examined. These pathways included an evaluation of aerobic (oxidative phosphorylation, Kreb's cycle and respiratory chain) and anaerobic metabolic capacity, hexose-monophosphate shunt activity, amounts of deoxyribonucleic and ribonucleic acids present and activity of beta-oxidation of fatty acids. Our results indicate that normal coronary arteriolar metabolism is predominantly aerobic. The findings also suggest a reduction in aerobic metabolism with an accompanying increase in anaerobic potential in the hyperthyroid coronary arterioles. Thus, during thyrotoxicosis, the coronary arterioles may partially shift from aerobic to anaerobic metabolism. Moreover, in both the normal and thyrotoxic rat heart, the coronary microvasculature appears quite stable with little cell proliferation. In contrast, both the control and hyperthyroid rat coronary arteries appear to utilize primarily anaerobic pathways, while the control and hyperthyroid myocardium seem highly dependent upon aerobic metabolism. The tremendous reduction in glucose-6-phosphate dehydrogenase activity in hyperthyroid, when compared to normal coronary arteries and some larger arterioles, implies a reduced capacity for nucleic acid and protein synthesis in the test animals.