The origin of lipid phagocytes in the central nervous system: I. The intrinsic microglia.

The potential for the transformation of the normal microglial cell to a lipid phagocyte was studied by light and electron microscopy in the brains of rodents and by light microscopy only in primates. All were subjected to some form of hypoxia-ischemia and the microglial response was examined in regions of selective neuronal destruction (SND) so that infarction was deliberately excluded. In vivo perfusion-fixation was employed in all animals and light microscopic examination was carried out on paraffin- and sometimes celloidin-embedded material. Semithin plastic sections from several regions of the rodent brains were used for light microscopy but ultrastructural studies were confined to the hippocampus. In all animals the microglia were activated and transformed into rod cells exhibiting phagocytic properties but only a minority gave rise to lipid phagocytes. Blood vessels were normal in all animals and no hematogenous elements were identifed in the the parenchyma. As neuronal ghosts could be identified for up to 3 weeks it was concluded that the capacity of the microglia for phagocytic activity was limited.

The origin of lipid phagocytes in the central nervous system: II. The adventitia of blood vessels.

The source of the lipid phagocytes that occupy a cerebral infarct has been determined by light and electron microscopy in the brains of rodents and by light microscopy only in the brains of primates. The infarcts (in neocortex, hippocampus, and thalamus) were the consequence of hypoxia-ischemia of various types. Hemorrhagic infarcts were excluded. After in vivo perfusion-fixation, paraffin- and celloidin-embedded material was used for light microscopy. Semithin plastic sections from the neocortex and thalamus were studied with the light microscope and ultrastructural studies were confined to the same regions. In all animals after about 2 days there was evidence of phagocytic activity in the fibroblasts in the adventitia of the remaining large vessels and also proliferation by mitotic division. At 5-7 days fibroblasts appeared to migrate from the vessels in a semifluid or fluid milieu and to give rise to typical phagocytes. These increased in size and number but signs of degeneration became apparent after 10 days and they had largely disappeared by 32 days. Smooth muscle cells and pericytes showed evidence of degeneration and phagocytic activity was never seen in the latter.

Temporal profile of neuronal damage in a model of transient forebrain ischemia.

This study examined the temporal profile of ischemic neuronal damage following transient bilateral forebrain ischemia in the rat model of four-vessel occlusion. Wistar rats were subjected to transient but severe forebrain ischemia by permanently occluding the vertebral arteries and 24 hours later temporarily occluding the common carotid arteries for 10, 20, or 30 minutes. Carotid artery blood flow was restored and the rats were killed by perfusion-fixation after 3, 6, 24, and 72 hours. Rats with postischemic convulsions were discarded. Ischemic neuronal damage was graded in accordance with conventional neuropathological criteria. Ten minutes of four-vessel occlusion produced scattered ischemic cell change in the cerebral hemispheres of most rats. The time to onset of visible neuronal damage varied among brain regions and in some regions progressively worsened with time. After 30 minutes of ischemia, small to medium-sized striatal neurons were damaged early while the initiation of visible damage to hippocampal neurons in the h1 zone was delayed for 3 to 6 hours. The number of damaged neurons in neocortex (layer 3, layers 5 and 6, or both) and hippocampus (h1, h3-5, paramedian zone) increased significantly (p less than 0.01) between 24 and 72 hours. The unique delay in onset of ischemic cell change and the protracted increase in its incidence between 24 and 72 hours could reflect either delayed appearance of ischemic change in previously killed neurons or a delayed insult that continued to jeopardize compromised but otherwise viable neurons during the postischemic period.

Columnar alterations of NADH fluorescence during hypoxia-ischemia in immature rat brain.

Cerebral hypoxia-ischemia was produced in 7-day postnatal rats by unilateral carotid artery ligation combined with systemic hypoxia (8% O2). Levels of high energy phosphates, which were only slightly altered in the contralateral hemisphere, were nearly depleted in the ipsilateral hemisphere during the 3-h hypoxic insult. With hypoxia of between 1 and 3 hours' duration, columnar alterations of cortical NADH fluorescence occurred in the same location and regional pattern as did histologic damage demonstrated previously (Rice et al., 1981). In regions exhibiting columns of NADH fluorescence, there was no evidence of a columnar reduction of high energy phosphates as levels of ATP and phosphocreatine were nearly zero. Recovery from 3 h of hypoxia was accompanied by partial and regionally heterogeneous restoration of ATP within the ipsilateral hemisphere. Columnar variations of NADH fluorescence were not detected in the recovery period; rather, regions with impaired restitution of high energy phosphates exhibited NADH fluorescence that was diminished diffusely compared to the contralateral hemisphere. The correlation between depressed NADH fluorescence and depleted ATP, present as cortical columns during hypoxia and as larger regions during recovery, suggests that decreased formation of NADH may be limiting the resynthesis of high energy phosphates.

The influence of immaturity on hypoxic-ischemic brain damage in the rat.

Brain damage in the Levine preparation (unilateral common carotid artery ligation with hypoxia) consists of ischemic neuronal alterations in the ipsilateral forebrain. As the model has been restricted to adult animals, unilateral common carotid artery ligation was carried out in 7-day-postnatal rats. Four to 8 hours later the 25 pups were exposed to 8% oxygen at 37 degrees C for 3.5 hours. Controls consisted of littermates subjected to carotid ligation without subsequent hypoxia, hypoxia without prior ligation, and neither ligation nor hypoxia. After hypoxia the animals were returned to their dams and appeared normal for up to 50 hours. All pups were then killed by perfusion-fixation. Moderate to severe ischemic neuronal changes were seen in the ipsilateral cerebral cortex, striatum, and hippocampus in at least 90% of the animals and included infarction in 56% of the brains. Cortical damage was occasionally laminar but more often occurred in columns at right angles to the pial surface. Unlike adult animals, there was necrosis of white matter, greater ipsilaterally, originating in and spreading from myelinogenic foci. The evolution of ischemic cell change and the associated gliomesodermal reaction was more rapid than in the adult. In 22 additional pups subjected to carotid artery ligation and hypoxia, brains were analyzed for water content. Significant increases (0.6 to 3.3%) in water content of the ipsilateral hemispheres occurred in 11 of 22 brains (50%). Unilateral ischemia combined with hypoxia in developing rats therefore results in neuronal destruction in the same brain regions as in adult animals, but also causes necrosis of white matter. The incidence of increased water content was similar to that of overt infarction. Thus, as previously shown in the adult, brain edema is a consequence rather than a cause of major ischemic damage in the immature animal.

The pathogenesis of ischaemic neuronal damage along the cerebral arterial boundary zones in Papio anubis.

The pathogenesis of ischaemic neuronal damage along the arterial boundary zones of the forebrain was investigated in 20 lightly anaesthetized, spontaneously breathing baboons. A combination of bilateral common carotid artery occlusion and systemic hypoxia was used. An arterial PO2 of 21.2 +/- 2.5 mmHg was maintained for about 20 min. Additional occlusion of the left common carotid artery for 20 min had no effect on the EEG (except for one animal with a cerebrovascular anomaly). Only when occlusion of the right carotid artery was added did the EEG become almost or completely isoelectric after an interval ranging from 23 s to 44 min (sequential common carotid artery occlusion while breathing air did not affect the EEG). After a chosen period of electrical silence, hypoxia and carotid occlusion were terminated. Hypotension did not occur during carotid occlusion or the recovery period. Survival was deliberately limited to 46 h, during which neurological assessment was made and the EEG was recorded just before in vivo perfusion-fixation of the brain. Neurological deficits included asymmetrical quadriparesis and myoclonus epilepsy. The brains of 3 animals were normal and in the 15 with brain damage this was restricted in the cerebral cortex to the arterial boundary zones. In the presence of profound hypoxia the oligaemia due to bilateral carotid occlusion can reduce tissue oxygenation locally to a level critical for the production of ischaemic damage in the cortical boundary zones. Portions of the basal ganglia were also involved in 7. The quantified brain damage scores correlated with the EEG scored on a six-point scale during the perod of electrical silence and early recovery. Brain damage scores also correlated with the times for intracranial pressure to return to normal levels from the peaks recorded just after the end of arterial occlusion and hypoxia. As brain damage only occurred when the EEG during bilateral carotid occlusion and hypoxia was silent for at least 8 min, it was concluded that in a variety of clinical settings a simple EEG-based monitoring system would be optimal for the detection of an impending failure of cerebral oxygen supply.

A new model of bilateral hemispheric ischemia in the unanesthetized rat.

A new model of transient, bilateral hemispheric ischemia in the unanesthetized rat is described. During ether anesthesia the rat's vertebral arteries were electrocauterized through the alar foramina of the first cervical vertebra and reversible clasps placed loosely around the common carotid arteries. Twenty-four hr later, the awake rats were restrained and the carotid clasps tightened to produce 4-vessel occlusion. The carotid clasps were removed after 10, 20 or 30 min of 4-vessel occlusion and the animals killed by perfusion fixation 72 hr later. Rats which convulsed during the ischemic or post-ischemic period were excluded from further study. All rats subjected to 20 or 30 min of 4-vessel occlusion demonstrated ischemic neuronal damage. The H1 and paramedian hippocampus, striatum and layers 3, 5 and 6 of the posterior neocortex were the regions most frequently damaged. The advantages of this model are the ease of preparation of large numbers of animals, a high rate of predictable ischemic neuronal damage, a low incidence of seizures and the absence of anesthesia.

Selective chromatolysis of neurons in the gerbil brain: a possible consequence of "epileptic" activity produced by common carotid artery occlusion.

Unilateral (50 to 118 minutes) and bilateral (2 to 33 minutes) carotid artery occlusion in gerbils resulted in two distinct types of neuronal alteration: ischemic cell change (ICC) in selectively vulnerable brain regions, and selective chromatolysis (SC) confined to the deeper layers of the cortex, the Sommer sector of zone h-1, and the paramedian region (PM) of the hippocampus. In typical SC the nucleus was eccentric and the Nissl substance was lost in the central eosinophilic cytoplasm. In electron micrographs this area of cytoplasm showed disruption of smooth and rough endoplasmic reticulum with disaggregation of polyribosomes and accumulation of mitochrondria and various dense bodies. SC was identified at 2 to 3 hours and was still recognizable at five days. When bilateral carotid artery occlusion lasted 5 to 6 minutes, SC was seen in the hippocampal Sommer sector and cerebral cortex, while ICC was restricted to the endfolium (h3-5). Unlike ICC, the frequency of SC was not related to the duration of ischemia but probably to the epileptic seizures (overt and subclinical) initiated by ischemia in the gerbil. These changes must be considered when the gerbil is employed as a model of experimental stroke.

Delayed pentobarbital administration limits ischemic brain damage in gerbils.

The capacity of delayed barbiturate administration to limit brain damage after unilateralcerebral ischemia was examined histologically in gerbils. The right common carotid artery was occluded in 50 animals under brief (3-minute) halothane anesthesia; 18 animals (36%) developed motor abnormalities consistent with stroke. The arterial clasps were removed after 1 hour and the abnormal animals were divided into treatment and placebo groups. Treated gerbils received sodium pentobarbital (70 mg/kg) intarperitoneally 1 hour after clasp removal and a smaller dose (50 mg/kg) 2 hours later; these animals lost corneal reflexes but retained spontaneous respiration and were kept normothermic. Animals in the placebo group received equivalent volumes of normal saline. Except for the period of anesthesia, both groups had similar postischemic motor behavior. Neuropathological examination of animals killed by perfusion-fixation after 24 hours revealed fewer pentobarbital-treated animals with shift of midline structures and with ipsilateral ischemic damage (including infarction). Compared with the placebo group, there was less extensive neuronal ischemic cell change in five regions of the ipsilateral cerebral hemispheres of the pentobarbital-treated animals (p less than 0.05). The results suggest that barbiturates administered as long as 1 hour after the end of an ischemic insult can still limit brain damage.

Epileptic brain damage: the role of systemic factors that modify cerebral energy metabolism.

The possible role of systemic physiological changes (occurring secondarily during status epilepticus) in the causation of epileptic brain damage has been evaluated in rats. Animals were anaesthetized, paralysed and mechanically ventilated; sustained electrocortical seizure discharges were induced by the intravenous injection of bicuculline, 1.2 mg/kg. After two hours of seizure activity brains were fixed by perfusion for histology. Physiological variables were maintained within certain limits from the end of the initial seizure phase (approximate duration twenty minutes) until two hours after onset of seizure to provide six groups: (1) Standard: mean arterial pressure above 120 mmHg, no hypoxia or hypoglycaemia, rectal temperature close to 37 degrees C. (2) Moderate Hypotension: mean arterial pressure at 70-75 mmHg. (3) Severe Hypotension: mean arterial pressure at 50 mmHg. (4) Hypoxia: arterial oxygen tension at 50 mmHg. (5) Hypoglycaemia: non-fed animals, with blood glucose close to 3.0 mumol/g. (6) Hyperthermia: rectal temperature at 40 degrees C. Microvacuolation and ischaemic cell change were identified by light microscopy in scattered neurons in the cortex (principally in the outer layers) in animals in three groups (Standard, Severe Hypotension and Hyperthermia). Similar neuronal changes were seen in the hippocampus (predominantly in the h1 or Sommer sector) in the Standard and Hyperthermia Groups. It is tentatively proposed that neuronal damage in animals with unrestricted cerebral oxygen and glucose availability is due to oxidative mechanisms in cells with excessively enhanced neuronal activity and that lesions caused by failing energy production do not appear until severe degrees of hypoxia are reached.

E.E.G. monitoring for the control of anaesthesia produced by the infusion of althesin in primates.

The continuous infusion of Althesin under electroencephalographic (e.e.g.) control provided a constant level of light anaesthesia for periods of 1--5.5 h during experimental brain hypoxia in spontaneously breathing baboons and Rhesus monkeys. Polygraphic records (respiration, heart rate, arterial pressure, cerebral venous sinus pressure, end-tidal gas concentrations) and also estimation of blood-gas tensions, pH, and concentrations of pyruvate and lactate demonstrated a steady physiological state. Various methods of e.e.g. monitoring were tested to establish an optimal assessment of depth of anaesthesia as a guide to the control of the rate of infusion of Althesin. A purpose-built modification of the Cerebral Function Monitor was found to give unequivocal recognition of changing depths of anaesthesia.

Profound hypoxia in Papio anubis and Macaca mulatta--physiological and neuropathological effects. I. Abrupt exposure following normoxia. II. Abrupt exposure following moderate hypoxia.

Lightly anaesthetized and spontaneously breathing P. anubis (PA) and M. mulatta (MM) inhaled at ambient pressure 3.2% oxygen (identical to 37,500 ft or 11,430 m) from air and also after pre-exposure to 14% oxygen (identical to 10,000 ft or 3,048 m). The EEG, ECG, respiratory rate, arterial and cerebral venous sinus pressures, end-tidal pO2 and pCO2 and body temperature were recorded. Arterial and cerebral venous sinus blood gases, pH and pyruvate and lactate contents were estimated. Before hypoxia, MM showed a relative hyperventilation. Profound hypoxia, from air, ended with the "last breath" at 89--205 sec in PA and at 93--570 sec in MM. Brain damage was restricted to one MM (4 exposures). Profound hypoxia after exposure to 14% oxygen ended with the "last breath" at 87--210 sec in PA and at 120 sec--94 min (including 9 exposures over 5 min) in MM. Brain damage was restricted to one MM ("last breath" at 94 min). In the two MM with brain damage there was evidence of reduction in cerebral perfusion near the end of profound hypoxia. Brain damage in one animal contrasts with the frequent and often severe brain damage in MM after equivalent sub-atmospheric decompressions preceded by exposure to moderate altitude (10,000 ft).

Reversible profound depression of cerebral electrical activity in hyperthermia.

Transient major reduction of EEG activity in an hyperpyrexic patient (rectal temperature 42.5 degrees C) and transient isoelectric ECoG during accidental hyperthermia (rectal temperature 41.8 degrees C) in a Rhesus monkey are reported. Since recovery of electrocortical activity occurred in both instance this implies that in hyperthermia, as well as in hypothermia, an isoelectric EEG may not indicate irreversible brain damage.