Depression of acetylcholinesterase synthesis following transient cerebral ischemia in rat: pharmacohistochemical and biochemical investigation.

The effect of transient cerebral ischemia on acetylcholinesterase (AChE) synthesis was studied in rats by a modified pharmacohistochemical method. The procedure involved in vivo irreversible inhibition of AChE by administration of the inhibitor diisopropyl fluorophosphate (DFP; 1.2 mg/kg b.w., i.m.) 1 h before 30 min forebrain ischemia (the four-vessel occlusion model). At the onset of ischemia, 70-75% of AChE was inhibited in the brain. Recirculation was followed by histochemical and biochemical investigations of newly synthesized AChE in the striatum, septum, cortex and hippocampus. Control sham-operated animals were treated with the same dose of DFP. For correlation, rats not treated with DFP were subjected to the same ischemic procedures and investigated simultaneously. In these rats, significant decrease in AChE activity was found in the striatum, septum and hippocampus during 24 h recirculation. In DFP treated rats, ischemia markedly depressed resynthesis of AChE; after 4 h recirculation, AChE activity was decreased by 45-60% in all investigated areas in comparison with controls and the AChE histochemistry showed only slightly stained neurons in the striatum and septum. Twenty-four hours after ischemia, these neurons were densely stained and the increase in AChE activity indicated a partial recovery of the enzyme synthesis. These results suggest that the depression of AChE synthesis after forebrain ischemia is probably transient, not accompanied by cholinergic neuron degeneration.

Neovascularization of the dorsal root ganglia transplanted into the olfactory bulb of neonatal rats.

Dorsal root ganglia (DRG), whose vessels are permeable to blood-born proteins, were syngenically transplanted into 2-5 days old Wistar albino rats after partial unilateral bulbectomy. Our aim was to follow survival and vascularization of the DRG, transplanted into a new environment, the developing olfactory bulb (OB). Three months after grafting the DRG graft was found fused with the spared portion of the OB. Only a subpopulation of the transplanted neurons survived the transplantation. Cholinesterase histochemistry showed BuChE positive vessels of host origin around the surviving DRG neurons. The majority of the vessels was impermeable to i.v. applied fluorescent dyes (Evans blue and lucifer yellow) and only few (1-3 vessel profiles/section) of them were labeled at the graft surface. By lanthanum nitrate tracing at the ultrastructural level, tight junctions were seen in the majority of the blood vessels of the graft. Our study shows that during the neovascularization the transplanted DRG is invaded by the host-derived blood vessels which possess blood brain barrier properties-they are impermeable to applied micro-and macromolecules. The newly formed/reestablished circulation appeared to be sufficient for maintaining a subpopulation of the transplanted sensory neurons.

Epidural perfusion cooling protects against spinal cord ischemia in rabbits. An evaluation of cholinergic function.

The protective effect of regional epidural spinal cord cooling was evaluated in a rabbit spinal cord ischemia model. Hypothermia was performed by the continual perfusion of 2-4 degrees C cold saline in the epidural space around the ischemic lumbar segments, 4 min before and during ischemia. The spinal cord was deeply hypothermic (21 degrees C) throughout the whole ischemic period. Ischemia was induced by the occlusion of the abdominal aorta for 40 min under normothermic or hypothermic conditions. Recovery of motor and sensory functions, spinal cord-evoked potentials, and motor-evoked potentials were then evaluated up to 24 h postischemia. After this period, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were measured, in particular, zones of the lumbar spinal cord. AChE was also investigated histochemically. Animals in the normothermic group displayed fully developed spastic paraplegia with near complete loss of spinal somatosensory and motor-evoked potentials. AChE histochemistry showed extensive necrotic changes affecting lumbosacral gray matter. These changes corresponding with the pronounced losses of ChAT and AChE activities indicated irreversible injury of the spinal cord. In contrast, after hypothermic ischemia, animals survived without any sign of neurological impairment with almost full recovery of the spinal cord-evoked potentials. ChAT and AChE activities in the gray matter showed near control values corresponding with histochemical analysis of fully preserved gray matter. Hypothermia under the present experimental conditions efficiently protected the spinal cord against ischemic injury.

Cholinergic enzymes in spinal cord infarction. Biochemical and histochemical changes.

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the ventral and dorsal horns and the intermediate zone of the rabbit lumbar spinal cord (L4-7) 24 and 96 h after ischemia caused by 20 or 40 min occlusion of the abdominal aorta. Changes of AChE and butyrylcholinesterase (BChE) activities were also detected histochemically by the direct thiocholine method. No significant changes were found immediately after ischemia. The most remarkable change after 20 min ischemia and 1 or 4 d of reperfusion was heterogeneous decrease in ChAT and AChE activities in the examined parts of gray matter. The highest loss of enzyme activities was found in the ventral horns and the lowest in dorsal horns. Following 40 min ischemia and reperfusion the significant depletion in enzyme activities in all investigated zones of the gray matter was accompanied with necrotic degenerative changes. There was a relatively greater decrease in ChAT and AChE activities in the ventral horns that corresponded with a more prominent morphological damage of the cholinergic neurons in this zone of the spinal cord.

Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study.

We have transplanted encapsulated dorsal root ganglia (DRG) from adult Wistar albino rats unilaterally into partially bulbectomized (n = 20) neonatal (P3-5) rats of the same strain. Three months postoperatively the animals were perfused and their brains processed by direct thiocholine method for cholinesterases (Ch), specific acetylcholinesterase (AChE) and nonspecific butyrylcholinesterase (BuChE) or stained by Cresyl violet. Selected sections were immunohistochemically stained for olfactory marker protein (OMP). In 17 cases we found surviving transplanted DRG. Fifteen transplants were well integrated with the spared portion of the olfactory bulb (OB) as clearly demonstrated by AChE and BuChE histochemistry, while two did not integrate. Regenerated OMP positive olfactory axons originating from neuroepithelium and AChE positive fibres from OB remnant penetrated into the transplants. In one case, fibers connected with BuChE positive Schwann cells grew from the transplanted DRG into the host OB. Individual sensory neurons of the transplants revealed variable intensity of the AChE staining, thus resembling the pattern of AChE activity in normal DRG. BuChE activity was mostly localized on the surface of sensory neurons in the ring of satellite cells. Some BuChE positive blood vessels penetrated into the DRG, and were observed around sensory neurons. The results showed a considerable viability and adaptability of the sensory neurons in the new environment after a long-term transplantation.

Effect of nerve growth factor on lesioned PC12 cells.

The protecting effect of nerve growth factor (NGF) from hydrogen peroxide was studied on PC12 cells conditioned at 1 mM hydrogen peroxide with NGF and without NGF in comparison with cells treated with neither hydrogen peroxide nor NGF. NGF treatment of PC12 cells increased significantly the activity of catalase representing induction of free radical detoxifying mechanisms. The protection effect of NGF was reflected also on enhanced activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the cells.

The influence of mechanical injury on the metabolic activity of transplanted cerebral cortex.

The authors studied the metabolic activity of rat embryonic cerebral cortex grafts (ED 15-16) implanted into rat brains immediately (TR0) and 14 days (TR14) after cavity formation. Over a period of two months, the ATP, lactate and glucose concentration in TR0 transplants remained at the same level as observed in the intact cortex, whereas in TR14 transplants the ATP and glucose concentration fell significantly and the lactate concentration rose. The DNA concentration rose in both types of transplants, but the increase was more pronounced in TR0 grafts. Choline acetyltransferase activity (a neuron marker) fell significantly in both cases, but the decrease was greater in TR14 transplants. The results indicate that grafts implanted into the brain immediately after cavities had been formed have better metabolic activity and are capable of longer survival than grafts implanted 14 days after cavitation.

Effect of spinal cord ischemia on axonal transport of cholinergic enzymes in rabbit sciatic nerve.

The fast axonal transport of acetylcholinesterase (AChE) and the slow transport of choline acetyltransferase (ChAT) were measured by the stop-flow ligation technique in the sciatic nerve of rabbits 6 and 24 h after ischemia performed by the occlusion of the abdominal aorta which lasted 40 min. Activities of these enzymes were also measured in punched samples of the spinal cord (L5-6). Results were correlated with those obtained from the sham-operated control group. Six h after ischemia, its only apparent effect was a different distribution of accumulated enzymes in the central nerve segments. Twenty-four h after ischemia, the transport of AChE was markedly depressed; proximodistal accumulation decreased by 68%, whereas enzyme activity in the intact contralateral nerve and in the ventral horns of the spinal cord was preserved. No effect of ischemia on the retrograde axonal transport of AChE was observed in this experimental model. Cytoplasmic ChAT is much more susceptible to necrotic degeneration than membrane-bound AChE; 24 h after ischemia its activity decreased significantly in all investigated parts of the sciatic motoneurones but the rate of slow axonal transport did not seem to be affected.

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.

Effect of partial ischemia and axotomy on activities of cholinergic enzymes in lower motor neurons of the dog.

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the ventral spinal cord, ventral spinal roots and in the central and peripheral stumps of the sciatic nerve transected under conditions of partial ischemia (produced by aortic ligation just below the renal arteries) were compared to those obtained under intact blood supply in time intervals 5, 10, or 15 days after surgery. The significant increase of ChAT activity in the central part of the sciatic nerve following 15 days of partial ischemia correlated with less significant elevation of ChAT in the ventral spinal cord. The changes of AChE activity were not significant during partial ischemia. ChAT in the peripheral stump of the sciatic nerve following 5 days of partial ischemia was preserved by 40% and AChE by 20% more than under normal blood supply. On the contrary, in the next 5 days interval losses of enzymes activity in the degenerating nerve were greater. ChAT was almost totally inactivated whereas 50% of AChE activity was preserved until the end of period examined.

Choline acetyltransferase and acetylcholinesterase in canine spinal ganglia: increase of choline acetyltransferase activity following sciatic nerve lesion.

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in the dorsal spinal ganglia, the dorsal spinal root and the spinal cord of the normal adult dogs and following one side transection of the sciatic nerve in the intervals 5, 10, 15 and 21 days respectively. In the spinal ganglia of normal dogs very low ChAT activity was found; it was three orders lower than AChE activity. Within 5-10 days after the nerve section ChAT activity increased almost five times in the spinal ganglia while AChE activity remained without any changes. The elevation of ChAT activity correlated with that in the dorsal roots at 15th day and in the dorsal spinal cord at 21st day after the nerve section. Histochemical "direct-colouring" thiocholine method showed AChE-positive cells were distributed mainly in the peripheral area of the spinal ganglia. The spinal ganglion cells ranged from intensely AChE-positive to AChE-negative without correlation between cell size and AChE activity. The ChAT activity changes were evaluated in correlation to the cholinergic function in the spinal ganglion neurons.

The dynamics of choline acetyltransferase and acetylcholinesterase changes in dog spinal cord during ischemia.

Activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were measured in several regions of the spinal cord, spinal ganglia and the sciatic nerve following ligation of the abdominal aorta just below the renal arteries for 20; 40; 80 and 120 min. Aortic ligation for 40 min produced a significant increase in the activities of both enzymes in the lumbar spinal cord. After ligation lasting 80 min the activity of CAT dropped under the control level and that of AChE remained elevated and returned to the control level in animals sacrificed 120 after the ligation. Similar but smaller AChE elevation was also found in the lower thoracic and the sacral spinal cord, respectively. In ischemic spinal ganglia and the sciatic nerve a decrease in AChE activity was found.

Cholinergic enzyme activity during partial brain ischemia in the dog.

Activity of cholinacetyltransferase (ChAT. EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was monitored during occlusion of arteria cerebri media dx. (MCA) in five areas of the brain cortex, in nucleus caudatus and in the thalamus of the ipsilateral and contralateral hemisphere. After 1 hour of MCA occlusion ChAT and AChE activity was reduced in the ischemised region of the hemisphere, i. e. in gyrus ectosylvius anterior and gyrus sylvius anterior, whereas after 4 hours of occlusion the differences were not significant. In nc. caudatus and thalamus the activity of enzymes during ischemia did not change much.

Distribution of choline acetyltransferase and acetylcholinesterase in the nervous system of the dog.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were determined in 23 selected parts of the dog CNS and 4 parts of the peripheral nervous system. Maximum ChAT activity was found in the caudate nucleus and the ventral roots of the spinal cord. High activity was also present in the thalamus, the pons, the cerebral cortex, the medulla oblongata, the ventral spinal horns and the sciatic nerve. The lowest activity was measured in the cerebellum, the dorsal cord roots and the spinal ganglia. Maximum AChE activity was found in the caudate nucleus and the cerebellum. Relatively high activity was also present in the thalamus, the pons, the medulla oblongata, the grey matter of the spinal cord and the spinal ganglia. The lowest AChE activity was measured in the ventral and dorsal spinal roots.

Effect of dexamethasone on pituitary-adrenocortical response to acute irradiation stress in rats.

After the irradiation with a lethal dose of 387 mC kg-1 (1500 R) an increase of plasma and adrenal corticosterone concentration was found in male rats (about 250 g body weight) during first 24 h as compared to sham-irradiated animals, the maximum increase being observed at 60 min after irradiation. A second period of corticosterone increase was found at 48 and 72 h. At the same time the adrenal weight increased and severe lesions of gastrointestinal tract were found. In a second experiment dexamethasone (50 mg kg-1) was injected 3 h before irradiation and this prevented the increase of plasma and adrenal corticosterone level at 60 min after irradiation. It was suggested that the degradation of corticosterone might be slowed down during the first period after irradiation, while at the same time the feed-back regulatory mechanism of ACTH release from the pituitary apparently remained intact. The second period of corticosterone increase was apparently due to severe gastrointestinal lesions.

Changes in rat liver and adipose tissue lipogenesis in the course of continuous gamma radiation.

Lipogenesis in liver of continuously irradiated rats (dose rate of 0.57 Gy per day for up to 25 or 30 days) was increased, in comparison with control animals, between 7 and 30 days. In the liver of fed rats it was approximately five times higher than animals fasted for 16 hours before sacrifice. Lipogenesis in adipose tissue was decreased on the third day of irradiation and its value was higher than in controls between 7 and 25 days. Glyceride glycerol synthesis in irradiated rats was increased compared with controls on the first, seventh and between 21 and 25 days of continuous irradiation. We suggest that the increase in lipogenesis in the liver of continuously irradiated rats is an important factor in lipid metabolism disorders caused by the continuous irradiation.

Effect of ischemia on acetylcholinesterase activity and its molecular forms in the dog spinal cord, spinal ganglia and sciatic nerve.

Partial ischemia of the spinal cord was produced by the ligature of the abdominal aorta twice for 40 minutes with a 40 min. recirculation interval. At the end of the ischemic period acetylcholinesterase (AChE. EC 3.1.1.7) activity increased significantly in the lumbosacral part of the spinal cord but simultaneously decreased in the spinal ganglia; There was no significant change in the sciatic nerve. After 48 hours of recovery AChE activity returned to normal in the sacral part of the spinal cord and in the spinal ganglia, but a less expressive increase remained in the lumbal part. Electrophoretic separation of AChE on the polyacrylamide gel gave in intact control dogs: three or four molecular forms in the spinal cord, five in the spinal ganglia and two molecular forms in the sciatic nerve. Ischemia increased the relative content of the lower molecular forms and often a new low molecular form appeared.

Acetylcholinesterase activity and its molecular forms in selected regions of the dog's nervous system.

Acetylcholinestrase (AChE, EC 3.1.1.7) activity and its molecular forms were studied in the cerebral cortex, basal ganglia, thalamus, cerebellar cortex, medulla oblongata, the various parts of spinal cord, the spinal ganglia and the sciatic nerve in intact adult dogs. The number and the proportion of different molecular forms of AChE in these regions of the dog's nervous system varied considerably. The highest AChE activity was found in the basal ganglia, where only one molecular forms was detected. Activity was also high in the cerebellar cortex (2 isoenzymes), the thalamus (4 isoenzymes) and the medulla oblongata (5 isoenzymes), and was lower in the cerebral cortex (3 isoenzymes). AChE activity in the spinal cord was highest in the sacral part (4 isoenzymes); in the other parts (3 isoenzymes) it was equal. Five molecular forms were found in the spinal ganglia and two in the sciatic nerve. The significance of the multiple molecular forms of AChE is discussed.

Plasma and adrenal corticosterone levels in male rats following x-irradiation at different times of day.

Adrenal and plasma corticosterone levels were investigated in male rats irradiated at 3-h intervals during the 24-h period (light : dark - 12 h : 12h). Adrenal and plasma corticosterone levels were markedly increased in animals exposed to a single whole-body X-irradiation with a lethal dose of 387 X 10(-3) C/kg (1 500R) and a 24-h fasting, compared to the sham-irradiated fasting controls, even during the lowest values of the intact group (08.00 h); the difference between the mean values of the irradiated and control group was not significant. The basal diurnal rhythm of corticosterone was retained even after irradiation. In comparison with the control, sham-irradiated group, the animals exposed to irradiation with a non-lethal dose of 64.5 X 10(-3) C/kg (250R) and a 48-h fasting showed adrenal corticosterone levels insignificantly higher in the light phase and lower in the dark phase. In comparison with the intact controls, both irradiated and sham-irradiated groups showed considerably shifted fluctuations during the 24-h period: the lowest levels in the middle of the dark phase and the highest in the middle of the light phase. Plasma levels of corticosterone were lower in irradiated animals than in the controls throughout the 24-h period. There were more irregular fluctuations and the diurnal rhythm was disturbed.