Resuscitation 7, 263-270

Histochemistry of the post-resuscitation changes in the brain

GALINA N. MIROTVORSKAYA Research Laboratory of General Reanimatology, Academy of Medical Sciences of the USSR, str. 25th Oktyabrya 9, 103012 Moscow 12, USSR.

Summary In 20 dogs, acid mucopolysaccharides, gangliosides, lysosomal enzymes and iontransport enzymes were investigated. Material was taken 2,24 and 72 h after 15 min of clinical death due to electrotrauma. Changes in the post-resuscitation period indicated deterioration of transmembrane ion-metabolite transport in the brain. These changes had phasic variations that reflect the complexity of post-resuscitation processes. Introduction Knowledge of changes in cerebral tissue during the post-resuscitation period is incomplete and fragmentary, especially at the level of light-microscopic histochemistry. Nevertheless, even these incomplete data indicate that it is a pathological process with its clear dynamics in time. Cerebral ischaemia is one of the aetiological factors of this process. However paradoxical it may seem, the recovery itself is another factor for the renewal of cell activity promoting the development of further changes. Investigation of early and late post-resuscitation periods is important for the understanding of the dynamics and character of metabolic disorders. The aim of the present work was to study changes in chemical components localized mostly in the membrane, such as acid mucopolysaccharides, sialic acids of gangliosides, alkaline phosphatase, acetylcholine esterase (AChE), Na+-K+-ATPase and acid phosphatase (the lysosome marker), taking as a basis the biochemical studies of disturbances of active transport of ions in cerebral tissue during anoxia and ischaemia (Reneau, Zeuthen, Dora, Silver & Guibeau, 1975; Krnjevic & Morris, 1975; Rossowska, Lewandowski and Dabrowiedi, 1975; Kogure, Busto, Kishikawa & Scheinberg, 1976). Material and methods Twenty mongrel dogs of both sexes weighing 9-14 kg were resuscitated by the method of Negovsky (1966) after 4 and 15 min clinical death induced by an electric current. Control animals were killed both by electric current and by perfusion of isotonic solution of formalin under thiopental narcosis. The experimental dogs were killed by electric current, 2,24 and 72 h after resuscitation. The brain was excised not later than 5 263

264

G. N. MIROTVORSKAYA

POST-RESUSCITATION

BRAIN HISTOCHEMISTRY

265

min after death. Six areas of the brain, the motor cortex, hippocampus, amygdala, hypothalamus, the cerebral cortex and medulla oblongata, were examined. The following parameters were measured by the techniques described by the authors indicated: acid mucopolysaccharides (Hale); sialic acids of gangliosides (Mowry, described by Mogilnaya, 1966), alkaline and acid phosphatase (Gomori), AChE Holmstedt), Na+-K+-ATP-ase (Padikula-Hermann). All these methods are as described by Pearse (1962). Results

Acid phosphatase may be considered one of the most sensitive indexes of metabolic changes in the post-resuscitation periods. Its activity changed significantly even after 4 min of systemic circulatory arrest for 2 h, whereas reactions of the other activities under study did not change. Widespread diffusion of the reaction product was found in the cytoplasm of Purkinje cells, indicating a considerable rise of permeability of lysosomal membranes, with simultaneous significant increases of the number of neurons containing numerous large particles of the reaction product, which was due to an activation of lysosomes. Systemic circulatory arrest for 15 min led to a considerable rise in number of neurons with diffuse activity of the acid phosphatase, especially in the cerebral cortex and cerebellar cortex, in hippocampus (Fig. 1) and in the nuclei of the brain-stem reticular nuclei. This diffuse reaction indicated the penetration of lysosomal enzymes into the cytoplasm, due to increased permeability of lysosomal membranes. In the astrocytes, on the other hand, the activity of the enzyme was increased. The intensity of reaction to the acid mucopolysaccharides was decreased in the region of the cellular membrane of neurons and neuroglia and in the vessel walls. Control reactions indicated that this decrease was due to dermatan, chondroitin and heparin sulphates. The control degree of reaction remained in the pia mater and in the myelin. The reaction to the sialic acids was uniformly decreased along the periphery of neurons and neuroglial cell bodies in all of the cerebral sections studied. The activities of AChE, Na+-K+-ATPase and alkaline phosphatase were decreased in the cytoplasmic membranes of neurons and neuroglia, especially in layer V of the sensory-motor cortex, in the paraventricular and supraoptic nuclei of the hypothalamus and in the reticular formation of the brain stem. These changes were less pronounced in the vessel walls and in myelin. Hence, disturbances of ion-metabolite active transport already appeared during clinical death. Two hours after resuscitation from 15 min clinical death, the increase of permeability of the lysosomal membranes and the decreased reaction to sialic acids was as pronounced as at 15 min after circulatory arrest. However, the reaction to acid mucopolysaccharides and the activities of alkaline phosphatase, Na+-K +-APTase and AChE were decreased even more (Fig. 2). Twenty-four hours after resuscitation, the metabolic processes reverted to normal, in particular those of the permeability of cellular and vascular membranes. At the same time reparative processes were recorded. The reaction to acid mucopolysaccharides returned to approximately the control level. These changes were most distinct in neurons of the sensory-motor cortex, hippocampus and in the Purkinje cells. The acid phosphatase activity in neurons and neuroglia rose significantly; numerous

266 G. N. MIROTVORSKAYA

POST-RESUSCITATION

BRAIN HISTOCHEMISTRY

267

268

G. N. MIROTVORSKAYA

c

F

POST-RESUSCITATION

BRAIN HISTOCHEMISTRY

269

large particles of the reaction product appeared in the cytoplasm and in the cell processes. The permeability of lysosomal membranes remained very high only in the Purkinje cells. The activity of the alkaline phosphatase returned to the control level., The activity of Na+-K+-ATPase remained high in the cytoplasmic membranes and in the cytoplasm of neurons, especially in the nerve cells of layers III-V of the sensorymotor cortex, hippocampus, nuclei of the cranial nerves in the brain stem and in the Purkinje cells. The control enzyme activity was seen in neuroglia and in the vascular walls. Only the content of sialic acids and the AChE activity remained as low as they had been 2 h after resuscitation. However, after 72 h, despite the good apparent neurological recovery, signs of deterioration of the permeability in the cellular and vascular membranes appeared in the brain. Neurons with decreased reaction to acid mucopolysaccharides and with decreased activity of alkaline phosphatase (Fig. 3) and Na+-K+-ATPase (Fig. 4) predominated. The permeability of the lysosomal membranes’ weak homogeneous reaction in the cytoplasm was increased. Similar changes were found in the neuroglia, vascular walls and in the myelin. Discussion The analysis of the results led to the conclusion that passive diffusion increased in the brain in the post-resuscitation period. Thus the decrease of Na+-K+-ATPase activity indicated the disturbance of active transport of cations. The decrease of reaction to acid mucopolysaccharides indicated their depolymerization, which led to reinforcement of transport of substances including macromolecules through the cell membranes due to the concentration gradients. Ischaemia, even of short duration, caused changes in structural elements of the cerebral tissue, although if the period of circulatory arrest was very short, investigators could not find these changes under the microscope. Ischaemia induced in neurons and neuroglia different stages of functional renovation and destruction of their ultrastructures. It is extremely difficult for the cell to restore its metabolism and structure quickly if it is ageing rapidly at the time of ischaemia, although this state, near deterioration, is physiological. If the renovation of cellular ultrastructures has just terminated or is terminating, it is highly probable that its metabolism will accomplish more rapidly and adequately the reconstruction necessary to survive under postresuscitation conditions. The duration of ischaemia is in inverse proportion to the number of cells that can retain their metabolism and recover it quickly, for cells have metabolic excess allowing them to survive after deviations from their optimum function. The prolonged conservation and also the increase of ischaemic changes in the brain after resuscitation, led to the supposition that there exist special cellular mechanisms providing normal metabolism for some time and within certain limits. Their resources are not inexhaustible, which explains why, 72 h after resuscitation, the metabolic changes in the cerebral tissue may increase significantly. Perhaps the safety of these mechanisms and their durability are greater in the cells that were in the state of full differentiation at the onset of ischaemia. Thus histochemical changes in the brain reflect the complexity and the dynamics of post-resuscitation processes that combine the elements of restoration, compensation and the appearance of new pathological changes. These changes may be the result of decrease of pools of enzymes and structural

270 G. N. MIROTVORSKAYA

proteins. There is some indication in the literature that these resources cannot be replenished by nuclear or cytoplasmic synthesis in the post-resuscitation period (Albrecht & Yanagihara, 1978). References Albrecht, J. & Yanagihara, T. (1978) Effect of cerebral anoxia and ischemia on messenger RNA metabolism. Trans. Amer. Sot. Neurochem. 9, 147. Kogure, K., Busto, R., Kishikawa, H. & Scbeinberg, P. (1976) Suppression of neural activity in brain edema following stroke. In: Dynamic Aspects of Cerebral Edema, p. 32, Montreal. Krnjevic, K. & Morris, M. E. (1975) Strophanhidine effects on extracellular K+ and electrogenic pumping in the cuneate nucleus. J. Physiol (Land.) 250, 36. Mogilnaya, G. M. (1966) Histochemical detection of siahc acids. Arch. Pathol. 3, 77-78 (in Russian). Negovsky, V. A. (1966) Indirect Massage of the Heart and Expiratoric Artijicial Respiration, pps. 4761. Sovetskaya Rossiya, Moscow. (In Russian.) Reneau, D. D., Zeuthen, T., Dora, E., Silver, I. A. % Guibeau, E. J. (1975) An analysis of transient changes in brain ions following anoxia. In: Proc. 28th Ann. Co@ Eng. Med. and Biol., New Orleans, 17, p. 330. Rossowska, M., Lewandowski, W. & Dabrowiecki, Z. (1976/77) Effect of &hernia on the activity of (Na+Kf)-ATPase in the microsomal fraction of guinea pig brain. Bull. Acad. Pal. Sci., Ser. Sci. Biol. 24, 691-696.

Histochemistry of the post-resuscitation changes in the brain.

Resuscitation 7, 263-270 Histochemistry of the post-resuscitation changes in the brain GALINA N. MIROTVORSKAYA Research Laboratory of General Reanim...
3MB Sizes 0 Downloads 0 Views