Morphologic and cytophotometric indices of the state of the neurons of the spinal cord and spinal cord ganglia following exposure to gravitational stress]

The work presents data on the state of the neurons of the spinal ganglia and the spinal cord of the lumbosacral part in 96 cats subjected to single stresses (10g) and repeated ones (6g). The material was stained with thionin after Nissl. RNA was detected after Einarson. Photometry of the sections was made in MUP.5. Morphological changes were shown to be more pronounced in sensory cells. The method of cytophotometry established the increase by 25% amount of RNA in their cytoplasm after single stresses. Repeated stresses resulted in depletion of RNA. The amount of RNA returned to the initial level within 2 days after single stresses and within 3 days after repeated rotations. The shifts were much less pronounced in motoneurons.     

Influence of gravitational overload and hypokinesia on the structure of the vascular bed of the stomach]

Under study were effects of gravitation stresses, total hypokinesia and their combinations on blood vessels of the stomach. The work was carried out in 130 rabbits, 16 of them being used to study the normal structure of the gastric vascular bed. The vascular bed was injected with the Gerota's mass followed by clearing, making histological preparations and roentgenography. The investigation has revealed both quantitative and qualitative changes in the structure of the gastric blood vessels. The maximum endurable stress of the ventro-dorsal direction causes morphological shifts less pronounced than stresses of longitudinal directions. With prolonged terms of hypokinesia (1-12 weeks) morphological changes became more pronounced in all the layers of the stomach. A combination of successive gravitation stresses and hypokinesia during 4-12 weeks aggravated morphological changes which occurred after exposure to isolated above factors. The animals subjected to maximum endurable stresses before and after 4-week hypokinesia developed vascular changes more typical for the effects of gravitation. The pretraining of animals did not give a pronounced positive effect on the changes of the angioarchitectonics of all the gastric layers after a repeated stress following 4-week hypokinesia.     

State of Dogiel type II neurons in the wall of the cat small intestine following a single exposure to gravitational stress]

Under study was the nervous apparatus of the small intestine in 22 cats subjected to a single gravitation stress by rotation in a centrifuge of 1,5 m radius. The stress was equal to 10 units, duration from 2,5 to 3,5 min., and was of head-pelvis direction. The material was treated after Nissl and Gomori in Chilingarian's modification. Within 1-3 days after exposure to gravitation stresses in the Auerbach plexus there appeared changes in the shape of the body of the type II Dogiel cells, such as pericellular oedema, vacuoles in the cytoplasm, chromatolysis and varicous thickenings of nerve fibres. Terminal structures of the dendrites of the type II Dogiel cells are thickened. The changes are most pronounced within the 4th-7th days after exposure to gravitation and are observed as late as the 14th day, being sharply diminished by the 30th day.     

Effect of gravitational loads of a training nature on the blood vessels of the rabbit spinal cord]

Three series of experiments were performed in 47 rabbits. The cerebral vessels were studied by methods of injection and clearing. In the first series under study were vessels after exposure to stresses of a training type. The picture of the circulatory bed under these conditions was close to normal in morphological parameters (diameter of vessels, amount of visible vessels, distinctness, evenness of the contours pronouncement of networks etc). In the 2nd and 3d series the results of training were checked by a single exposure to a series of stresses or a solitary stress. The data obtained suggest increased resistance in majority of trained animals to the following hypergravitation and better morphological parameters of the spinal cord circulatory bed as compared with not trained animals. Hence, certain adaptation to the following hypergravitation can be obtained in rabbits by means of repeated rotations in a centrifuge according to a special schedule of training.     

Effect of transverse gravitational loads on the vessels of the retinal membrane of the rabbit eyeball]

Changes in the eye ball retina were studied in 3 series of experiments in cleared preparations after exposure of the animal to gravitation stress of transversal direction. Prolonged single functionally endurable stress of ventro-dorsal direction (10 units/3 min) caused dilatation of the retina vessels, worse filling of peripheral parts of the circulatory bed, uneven staining of the vascular wall, constriction and dilatation along the course of the vessel. More continuous exposure to the same value of stresses (10 units up to the animal's death) resulted in a pressor effect; qualitative changes in the vessels increased. Within 1,5 hour after a 3-minute-long exposure to stress the vascular bed still remained unchanged. The diameter of vessels approached normal while the general quantity of vessels was decreased and qualitative changes were well pronounced.     

Effect of gravitational loads in different directions on the structure of the lymphatic bed of the pelvic extremities in the rabbit in vivo]

The method of lymphorentgenography was used in order to study alterations in the lymph vessels of pelvic extremities of rabbits at different terms (from 1 week up to 6 months) after exposure to gravitation stresses of different directions, value and duration. After exposure to stresses of cranio-caudal direction the amount of vessels increased. They had weel pronounced sinuosity and clearly protruding valves. The diameter of the vessels was more than normal and was equal to 0,3-0,8 mm. Popliteal lymph nodes were also increased up to 12,6-6,7 mm. Within 6 months after stresses these changes retained. Stresses of caudal-cranial direction during all periods of observation (from 1 week up to 6 months) caused dilatation of vessels (0,3-0,6 mm), their sinuosity and enlargement of popliteal lymph nodes up to 10,5-6,2 mm. All these changes returned to normality within 6 months. Thus, most pronounced and resistant morphological changes of the lymphatic bed were caused by greatest endurable stresses of cranio-caudal direction.     

The regional changes of the catalytic NOS activity in the spinal cord of the rabbit after repeated sublethal ischemia

The regional distribution of catalytic NOS activity was studied in the lumbosacral segments of the spinal cord of the rabbit during single (8-min), twice (8-, 8-min) and thrice repeated (8-, 8-, 9-min) sublethal ischemia followed each time by 1h of reperfusion. Single ischemia/reperfusion induced a significant increase of cNOS activity in almost all spinal cord regions, with the exception of non-significant increase in the dorsal horn. Sublethal ischemia repeated twice produced a significant decrease of enzyme activity in the intermediate zone and ventral horn and an increase in the white matter columns. Within thrice repeated ischemia, the activity of cNOS in the gray matter regions was similar to that found after a single ischemia/reperfusion. For all the animals subjected to single and twice repeated sublethal ischemic insults, there was no neurological impairment. Following thrice repeated ischemic insults, four out of five of the experimental animals recovered only partially and one was completely paraplegic. Our results do not indicate a cumulative effect of repeated sublethal ischemia on cNOS activity and, consequently, on NO production. The NO generated during thrice repeated ischemia/reperfusion appears to have a detrimental effect on the neurological outcome.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and 5''-Nucleotidase in the Central Nervous System of a Myelin-Deficient Rat Mutant

2',3'-Cyclic nucleotide 3'-phosphodiesterase activity was examined in brains and spinal cords of normal and myelin-deficient Wistar rats. While the activity in normal brains increased from 0.2 mumol/min/mg protein (units) at 6-10 days to 3.5 units at 25 days of postnatal age, the activity in the myelin-deficient rat remained at 0.2-0.3 units over the same period. In spinal cord, the normal activities were 5.7 and 10.9 units at 12 and 20 days, respectively, whereas they declined in the myelin-deficient rat from 1.06 to 0.79 units for the same age points. 5'-Nucleotidase activities in brain and spinal cord were normal in the myelin deficient rat at both ages.     

Short-term changes of NADPH diaphorase-exhibiting neuronal pools in the spinal cord of rabbit after repeated sublethal ischemia

The aim of this study was the histochemical characterization of NADPH diaphorase-positive neuronal pools in the rabbit lumbosacral segments using a model of single, repeated and multiple sublethal spinal cord ischemia. Following a single 8-min sublethal spinal cord ischemia and 1-hour reperfusion, the staining of NADPH diaphorase-exhibiting neurons in the dorsal horn, pericentral region, dorsal gray commissure and sacral parasympathetic nucleus was comparable with the control sections. In contrast to the foregoing sublethal ischemia, a regionally different somatic NADPH diaphorase (NADPHd) staining was found after multiple sublethal spinal cord ischemia. Whereas an almost complete loss of the staining of large NADPHd-exhibiting somata in the pericentral region was detected, the staining of the NADPHd-exhibiting neuronal pools in the deep dorsal horn and sacral parasympathetic nucleus was fully preserved. Concomitantly, a prominent reduction of small NADPH diaphorase-positive neurons was noted in the superficial dorsal horn layers of lower lumbar and sacral segments.     

Limited depletion of central adrenaline stores following administration of adrenaline synthesis inhibitors in rats

The aim of the present study was to maximise the selective depletion of adrenaline stores in the rat brainstem and spinal cord, obtained by administering inhibitors of adrenaline synthesis. Partial depletions of adrenaline in the hypothalamus and medulla were observed after a single injection of either LY134046 or SKF 64139 (40 mg/kg i.p.). The rate and extent of depletion seen in the hypothalamus 3 or 6 h after a single dose of LY134046 could be increased by simultaneous exposure to cold or by lowering blood pressure, but not by prior administration of the synthesis inhibitor at 12 h intervals for 3 days. None of the treatments used were able to significantly lower spinal cord adrenaline levels, despite at least 80% inhibition of spinal cord PNMT activity. These results suggest that the ability to reduce central adrenaline stores by synthesis inhibition is limited, especially in the spinal cord.     

Histological effects of 6-mercaptopurine on the fetal rat central nervous system: a light-microscopic study.

Wistar rats were administered single doses of 16 or 50 mg/kg 6-mercaptopurine (6-MP) on day 12 of pregnancy. Necrosis in the fetal forebrain and spinal cord was studied by light microscope 6, 12, 14, 48, 72, and 81 h and 8 days afterward. The extent of necrosis was dose dependent. The first necroses were seen after 24 h, regardless of location (brain, spinal cord) or dose; but the extent was greatest after 48 h. All necrotic cells had a typical appearance; they were ballooned and often fragmented, their nuclei were darkly colored and frequently pyknotic, and they were often karyorhexic. Necroses appeared almost exclusively at sites of beginning cellular differentiation, i.e., in the intermediate zone. In the spinal cord the ventricular zone was also necrotic and the alar plate (dorsal horn) always affected. Phagocytizing cells (macrophages) appeared in the spinal cord after 48 h and in the brain after 72 h. After 81 h all the necrotic material had been phagocytized, at which time there was a massive congestion of the extra- and intracerebral vessels. Hemorrhages appeared in defined localizations. Eight days after exposure to 16 mg/kg 6-MP fetuses no longer showed any visible deviations. Fetuses exposed to 50 mg/kg showed deviations in the cytoarchitecture of the neopallium: an extremely broadened ventricular zone, few cells in the intermediate zone, and extensive rarefaction cells in the cortical plate with no clear layer structure. In the spinal cord, cleft formations were especially noticeable in the dorsal-horn region. All fetuses showed a hydrocephalus externus after 50 mg/kg. The mechanism leading to necrosis is discussed.     

Long-range afferents in the rat spinal cord. 1. Numbers, distances and conduction velocities.

The caudal extent of the penetration of primary afferent axons from the T12 and L1 dorsal roots and sural nerve has been investigated in adult decerebrate spinal rats. Microelectrode stimulation at the root entry zone (REZ) and at further caudal points in the spinal cord was used to generate antidromic action potentials in single fibres recorded in dorsal roots or peripheral nerves. A total of 209 units were recorded in T12 and L1 dorsal roots and 27% of these could be antidromically activated 10 mm caudal to the REZ. Fifteen percent of the units could be stimulated at the L4-5 border, 15 mm caudal to the T12 segment whereas 4.5% of the axons could be stimulated 25 mm caudally in the S4 segment, 11 segments caudal to the entry segment. Similar recordings made from units in the sural nerve showed that of all the sural axons that penetrated to the L6 segment 50%, 18% and 2% of these reached the S1, S2 and S4 segments respectively. The conduction velocities of these units were clearly in the A-beta range when recorded in the nerve but decreased on entering the spinal cord and were reduced by 83% at their caudal end point. The results show that substantial numbers of primary afferents have long-ranging caudal branches in areas beyond the regions of known postsynaptic effects. The functions of these caudal projections are unclear but they may represent a potential substrate for the development of functional connections under conditions of disease or denervation.     

Effect of axotomy on cholinergic enzyme activities in the spinal cord and sciatic nerve of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity measured in the ventral and dorsal part of the dog spinal cord (L6-S2) and in the stumps of the sciatic nerve 5, 10, 15 and 21 days after its transection were compared with the corresponding activities in the intact contralateral nerve and in sham-operated animals. AChE was also examined histochemically. Changes in the enzyme activities in the central nerve stump were correlated with activity changes in the spinal cord. In the central nerve stump, a marked (25%) increase in AChE activity was found on the fifth day after transection, but by the 21st day it fell below control value levels; up to the 15th day it showed good correlation with AChE activity in the ventral spinal cord. Histochemically, pronounced reduction of enzymatic activity was found in the ipsilateral part of the spinal cord. On the 15th day, ChAT activity in the ventral spinal cord was also significantly decreased and the accumulation of the enzyme in the central nerve stump was negligible. On the contrary, at the last 21-day interval examined, a significant increase in ChAT activity and a nonsignificant increase in AChE activity was found in the spinal cord, but their activities in the central nerve stump were decreased. In the degenerated peripheral nerve stump ChAT activity dropped by an average of 99% and AChE activity by 48% during the first 15 days after transection but, on the 21st day, AChE activity was 22% higher than at the preceding interval.     

Gender differences in oxidative stress in spinal cord of rats submitted to repeated restraint stress

Behavioral and neurochemical gender-specific effects have been observed following repeated stress. The aim of this study is to verify the effects of repeated restraint stress on free radical production (evaluated by DCF test), lipoperoxidation (evaluated by TBARS levels), and total antioxidant reactivity (TAR) in the spinal cord of male and female rats. Results demonstrate no effect on lipoperoxidation; chronic stress decreased TAR both in male and female spinal cord. In addition, gender differences were observed both in TAR and in the production of free radicals, both being increased in females. These results may be relevant to the gender-specific differences observed after exposure to repeated stress.     

Modification of radiation myelopathy by the transplantation of neural stem cells in the rat

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2-C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of (60)Co gamma rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 microl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 microl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.     

Time-related changes of motor unit properties in the rat medial gastrocnemius muscle after spinal cord injury. I. Effects of total spinal cord transection

The contractile properties of motor units (MUs) were electrophysiologically investigated in the medial gastrocnemius (MG) muscle in 17 Wistar three-month-old female rats: 14, 30, 90 and 180 days after the total transection of the thoracic spinal cord and compared to those in intact (control) rats. A sag phenomenon, regularly observed in unfused tetani of fast units in intact animals at 40 Hz stimulation, almost completely disappeared in spinal rats. Therefore, the MUs of intact and spinal rats were classified as fast or slow types basing on 20 Hz tetanus index, the value of which was lower or equal 2.0 for fast and higher than 2.0 for slow MUs. The MUs composition of MG muscle changed with time after the spinal cord transection: an increasing proportion of fast fatigable (FF) units starting one month after injury and a disappearance of slow (S) units within the three months were observed. In all MUs investigated the twitch contraction and half-relaxation time were significantly prolonged after injury (p < 0.01, Mann–Whitney U-test). Moreover, a decrease of the fatigue index for fast resistant (FR) and slow MUs was observed in subsequent groups of spinal rats. No significant changes were found between twitch forces in all MU types of spinal animals (p > 0.05). However, due to a decrease of the maximal tetanic force, a significant rise of the twitch-to-tetanus ratio of all MUs in spinal rats was detected (p < 0.01). The considerable reduction of ability to potentiate the force was noticed for fast, especially FF type MUs. In conclusion, the spinal cord transection leads to changes in the proportion of the three MU types in rat MG muscle. The majority of changes in MUs’ contractile properties were developed progressively with time after the spinal cord injury. However, the most intensive alterations of twitch-time parameters were observed in rats one month after the transection.     

A clonogenic survival assay of neural stem cells in rat spinal cord after exposure to ionizing radiation

Neural stem cells play an important role in neurogenesis of the adult central nervous system (CNS). Inhibition of neurogenesis has been suggested to be an underlying mechanism of radiation-induced CNS damage. Here we developed an in vivo/ in vitro clonogenic assay to characterize the survival of neural stem cells after exposure to ionizing radiation. Cells were isolated from the rat cervical spinal cord and plated as single cell suspensions in defined medium containing epidermal growth factor and basic fibroblast growth factor. The survival of the proliferating cells was determined by their ability to form neurosphere colonies. The number and size of neurospheres were analyzed quantitatively at day 10, 12, 14 and 16 after plating. Plating cells from 5, 10 and 15 mm of the cervical spinal cord resulted in a linear increase in the number of neurospheres from day 10-16. Compared to the nonirradiated spinal cord, there was a significant decrease in the number and size of neurosphere colonies cultured from a 10-mm length of the rat spinal cord after a single dose of 5 Gy. When dissociated neurospheres derived from a spinal cord that had been irradiated with 5 Gy were allowed to differentiate, the percentages of neurons, oligodendrocytes and astrocytes as determined by immunocytohistochemistry were not altered compared to those from the nonirradiated spinal cord. Secondary neurospheres could be obtained from cells dissociated from primary neurospheres that had been cultured from the irradiated spinal cord. In conclusion, exposure to ionizing radiation reduces the clonogenic survival of neural stem cells cultured from the rat spinal cord. However, neural stem cells retain their pluripotent and self-renewing properties after irradiation. A neurosphere-based assay may provide a quantitative measure of the clonogenic survival of neural stem cells in the adult CNS after irradiation.     

Microiontophoresis of 5-hydroxytryptamine, epinephrine, and prostaglandin E1 on spinal neurons in the frog.

5-Hydroxytryptamine (5-HT) and epinephrine were applied by microiontophoresis to single neurons in the isolated spinal cord of the frog. 5-HT depressed all but two of the responsive cells, whereas the response to epinephrine consisted exclusively of depression. 5-HT action was more marked than that of epinephrine on most cells. With either compound, responseve units were diffusely distributed throughout the tissue. While it was proven that prostaglandin E1 (PGE1) exerts a direct excitatory action on spinal neurons, no evidence of an antagonism between PGE1 and the monoamines was obtained. These findings provide additional support to the hypothesis that 5-HT and epinephrine are transmitters in the frog spinal cord. The possibility that PGE1 may 'modulate' the responsiveness of spinal neurons to the monoamines was not confirmed.