Karl A. Conger

The Contribution of Reoxygenation to Ischemic Brain Damage

This study examined the hypothesis that the level of postischemic reperfusion affects the severity of the resulting neuronal necrosis. In rats, tissue Po2% was monitored as an index of flow (reoxygenation) at four cortical sites by chronically implanted platinum electrodes. Twenty minutes of total global cerebral ischemia was followed by 30 min of reoxygenation. The level of reoxygenation was controlled to maintain the Po2 nearly constant at one or more of the cortical electrodes. Tissue from within 400 μm of each of 19 electrode sites among seven rats was evaluated histologically. There was a positive correlation between reoxygenation level and severity of neuronal damage. Perineuronal lucent halo formation, probably representing astrocyte foot process swelling, was negatively correlated with reoxygenation level. This study demonstrates that ischemic neuronal damage was aggravated by increased reoxygenation but that perineuronal swelling, as evidenced by halo formation, was somewhat ameliorated.

Mathematical simulation of cerebral blood flow in focal ischemia.

A computer model was developed to describe regional cerebral blood flow and tissue oxygenation with autoregulation during focal ischemia produced by occlusion of th middle cerebral artery (MCA). This steady state model described the distribution of blood flow in the cerebral arterial system including the circle of Willis as well as the pial arterial anastomoses, and included a simplified form of autoregulation based on the local control of pressure and flow in the pial and intracerebral arteries, respectively. Preliminary simulation studies with the model yielded the following results. Less effective autoregulation was predicted by the model at low blood pressure in focal ischemia. Passive dilatation of the pial vasculature produced a leftward shift in the autoregulatory curve. Simulations with occlusion of the MCA revealed the ultimate importance of the pial anastomoses in providing adequate blood and oxygen supply in the ischemic territories including the specially vulnerable lenticulostriate area. The volume of the ischemic (pO2 less than 1 mmHg) brain tissue in the MCA-cortex estimated by using a concurrent Krogh cylinder model was 50% when the pial anastomoses were 80 micrometers in diameter and the ischemic area disappeared at 170 micrometers diameter. With relatively small anastomoses (less than 200 m) the model demonstrated intracerebral steal during intracerebral vasodilation. Passive dilation of the pial arteries including the pial anastomoses caused the steal to disappear and to reverse. These results suggest that both autoregulatory shift and steal reversal can be explained by passive dilatation of the pial vasculature.

Pressure distribution in the pial arterial system of rats based on morphometric data and mathematical models.

The objective of the present work was a theoretical evaluation of pial arterial pressures in normotensive rats and spontaneously hypertensive rats based on the geometry and topography of the pial arterial system as well as on various topological models of the vascular trees. Pial branches of the middle cerebral artery in the diameter range of 30–320 μm were selectively visualized by corrosion compound, and the diameter and length of vascular segments were measured. The vessels were classified into branching orders by the methods of Horsfield and Strahler. The steady-state pressure distribution in the pial arterial system was calculated assuming that the flow at the bifurcations was partitioned in proportion to a given power of the diameters of the daughter branches (diameter exponent). The maximum number of vascular segments along the longest branch varied between 16 and 33. The mean branching ratio was 4.14 ± 0.23 (SD). The mean diameter of vessels classified into Strahler orders 1–5 were: 50 ± 12, 71 ± 19, 106 ± 22, 168 ± 22, and 191 ± 7 μm, respectively. The calculated pressure drop in the pial trees of normotensive rats was approximately twice as large in proximal orders 3 and 4 than in distal orders 1 and 2. The mean pressure in arteries of order 1 ranged from 54.4 to 58.4 mm Hg in the normotensive rat (input pressure: 83 mm Hg), and from 77.2 to 89.0 mm Hg in the spontaneously hypertensive rat (input pressure: 110 mm Hg). The coefficient of variation of terminal pressures in vessels of order I increased linearly with the mean pressure drop in the system. The coefficient of variation in terminal pressure had a minimum as a function of the diameter exponent in case of each pial tree. At its minimum, it was higher in all spontaneously hypertensive rats (10.1–22.9%) than in any normotensive rats (6.0–8.5%). The corresponding diameter exponents were in most cases lower in the spontaneously hypertensive rat (1.3–2.5) than in the normotensive rat (2.5–3.0). Topologically consistent models of the pial arterial network predicted significantly less variation in intravascular pressures than was obtained by direct calculations. More idealized models suggested the dependence of coefficient of variation in terminal pressure on the total number of vascular segments contained by the tree. All models predicted the existence of the minimum of coefficient of variation in terminal pressure in function of the diameter exponent. From these, we conclude that (a) a hemodynamic configuration of the pial arterial system resulting in the smallest variation in cerebral perfusion pressure may exist, and (b) the pial vascular structure of spontaneously hypertensive rat allows less homogeneous terminal pressure distribution than does that of normotensive rats.

Concomitant EEG, Lactate, and Phosphorus Changes by 1H and 31P NMR Spectroscopy During Repeated Brief Cerebral Ischemia

Pilots of high-performance aircraft are subject to transient loss of consciousness due to cerebral ischemia resulting from sudden high gravitational stress. To assess the effects of gravitational stress-induced blackout on cerebral metabolism and electrical function, we developed an animal model in which global cerebral ischemia is produced repeatedly at short intervals. Rats were prepared by ligation of subclavian and external carotid arteries and the right carotid artery was cannulated bidirectionally to measure circle of Willis and systemic pressures. Ischemia was induced by inflation of an occluder about the left carotid artery. Interleaved 31P and 1H NMR spectra were acquired on a 4.7-T Biospec system simultaneously with EEG recordings. We report results from 20 experiments of 30-min duration in which rats were subject to 30 1-min ischemia:reflow cycles of 10I:50R, 20I:40R, 30I:30R, and 40I:20R [numbers are seconds of ischemia (I) and reflow (R) during each 1-min cycle]. During ischemia the graded delivery of the ischemic insult permitted direct correlations between 2- to 5- and 7- to 20-Hz EEG activity and progressive changes in pH, lactate, ATP, phosphocreatine (PCr) and Pi. The best correlations were found between EEG activity and pH and PCr; correlation coefficients ranged from 0.93 to 0.95. A loss of EEG activity was observed without significant sustained energy loss in all but the most severe cycle.

Computer-Regulated Constant Pressure Ischemia in the Rat: The Animal Model

A system permitting computer control of partial ischemia in the normotensive rat brain was developed. Right carotid cannulation and bilateral subclavian artery occlusion made the input of blood to the brain dependent solely on left carotid artery flow. Perfusion pressure was controlled by partial compression of this artery with a balloon. The system can produce a range of partial ischemic states maintaining perfusion pressures from 4 to 20 mm Hg. The adequacy of the servo-control system was evaluated in greater detail at requested perfusion pressures of 7 and 12 mm Hg in 14 male Sprague-Dawley rats (300–450 g). Experimentally obtained cerebral perfusion pressures of 6.84 (SD = 0.25, n = 7) and 11.72 (SD = 0.89, n = 7) mm Hg, respectively, demonstrate the efficacy of the system. CBFs were concurrently measured at four separate bilaterally symmetric anatomic sites (cortex, hippocampus, thalamus, and substantia nigra). Significant intra- and interhemispheric differences were found to exist, with regional flows monitored ipsilaterally to the carotid balloon exceeding those of the opposite hemisphere. In summary, this acute model of cerebral ischemia permits control of perfusion pressure over the entire critical partial ischemic range.

Nitroarginine Reduces Infarction After Middle Cerebral Artery Occlusion in Rats

Neuronal production of nitric oxide (·NO) provides a common link between two seemingly independent mechanisms of brain injury—excitatory neurotransmitters and oxygen radicals. The connection results from the recent demonstration that glutamate stimulates neurons to produce nitric oxide (Garthwaite et al. 1988). Neurons produce nitric oxide by oxidizing arginine with a calcium-activated enzyme that is physiologically activated by the N-methyl-D-asparate (NMDA) subclass of receptors (Garthwaite 1991). Neuronal nitric oxide helps regulate local cerebral blood flow, contributes in synaptic plasticity (Nowak 1992), and may have a role in the normal development of brain (Gaily et al. 1990). Ischemia and hypoxia initiate events that mimic the normal physiological regulation of the NMDA subclass of glutamate receptors, which may overproduce nitric oxide whentissue is reperfused. Oxidative metabolism disrupted by ischemia will produce the oxygen radical, superoxide (O2 –), that reacts with the elevated concentrations of nitric oxide to form the destructive species peroxynitrite (ONOO–) and thereby exacerbating cerebral injury.

Nitric Oxide as a Mediator of Cerebral Blood-Flow, Synaptic Plasticity, and Superoxide-Mediated Brain Injury

We propose that excessive production of nitric oxide initiated by activation of glutamate receptors after cerebral ischemia potentiates free radical injury to the brain. Nitric oxide, produced by neurons and possibly astrocytes, helps regulate local cerebral blood flow and plays an essential role in synaptic plasticity and normal development of the brain. Nitric oxide itself is a weak oxidizing agent, but after reaction with superoxide (O 2 - ), it forms the strong and relatively long-lived oxidant peroxynitrite anion (ONOO-) (Beckman et al., 1990). Peroxynitrite is sufficiently stable even in the presence of physiological concentrations of glutathione and other cellular antioxidants to diffuse for up to several cell diameters. Depending on what peroxynitrite reacts with, it can produce oxidants with the reactivity of hydroxyl radical (HO⋅), nitrogen dioxide (NO2), nitronium ion (NO 2 + ), and possibly singlet oxygen. Thus, the conversion of nitric oxide to peroxynitrite and related secondary oxidants may provide a common link between glutamate and free radical-mediated injury. Glutamate antagonists and free radical scavengers can reduce infarct volume by approximately the same extent in rat middle cerebral artery occlusion models of stroke (Oh and Betz, 1991). Recently, nitroarginine, a competitive inhibitor of nitric oxide synthesis, has also been shown to reduce infarct volume in a mouse middle cerebral ischemia model (Nowicki et al., 1991).

A mathematical model of the intracerebral steal phenomenon in regional and focal ischaemia.

The objective of the present work was to mathematically estimate the extent and dynamics of intracerebral steal which may occur in response to cerebral vasodilation in regional and focal cerebral ischaemia. To this end, a spatially distributed mathematical model of regional cerebral blood flow (rCBF) was developed. The model contained a parallel system of intracerebral vascular resistances which were connected in series to a lumped extracerebral artery resistance and, for the focal ischaemia model, also a lumped pial collateral resistance. The rCBF was measured at 30 min of ischaemia in the following models: (1) bilateral carotid occlusion in spontaneously hypertensive rats (SHR), and (2) occlusion of the middle cerebral artery (MCA) in normotensive rats. The measured 3-dimensional rCBF data were used to set up the initial values of intracerebral resistance components. Cerebral vasodilation induced by inhalation of CO2 was simulated in the model by decreasing the values of both intracerebral and collateral resistance. Vascular responsiveness was specified to decrease with the ischaemic rCBF. In addition, a long term change in rCBF and resistance distribution was introduced to account for: (1) gradual rise in intracerebral resistance due to ischaemic oedema, and (2) adaptive decrease in collateral resistance. The following were predicted by the mathematical model. (1) At 60% maximum intracerebral dilatation a small intracerebral steal (5-10%) occurs at flow levels below 30-50 ml/100 g/min in both ischaemic models. (2) In focal ischaemia, the steal can be compensated by the 5% to 20% decrease in the collateral vascular resistance. (3) The rate of collateral adaptation overcomes the rate of intracerebral resistance rise and, therefore, eliminates the intracerebral steal after an adequately long period of time (on the order of a few hours). (4) An inverse steal effect can be demonstrated at the end of vasodilatation, provided that the time constant of collateral adaptation selected is longer (about 5:1) than the time constant of the intracerebral resistance rise. We conclude that the prediction of rCBF response to vasodilatation in cerebral ischaemia requires a knowledge of resting rCBF and of the response characteristics of both intracerebral and pial arterial segments.

The role of tissue acidosis in ischaemic tissue injury: the conpept of the pH integral

Cerebral cortical tissue pH was monitored with an extracellular glass electrode in 32 rats subjected to total global cerebral ischaemia produced by ligation of the basilar and carotid arteries with systemic hypotension for periods of 8 to 60 min. The totality of the ischaemia, and its duration were confirmed by monitoring with a brain tissue O2 electrode. Reperfusion was induced by hypertension and maintained thereafter to exclude delayed ischaemia during 3 h survival after which the rats were sacrificed by perfusion fixation. The severity of tissue pH change was varied by inducing hyperglycaemia in some of the rats. Quantitative counts were made of neurons demonstrating changes reflecting severe ischaemic injury within 500 microns of the electrode tip. For the criterion of an ischaemically injured neuron count greater than 20%, there appeared to be a threshold at about 30 min, and more than 0.8 units change in pH. For quantitative assessment of the ischaemic insult a more satisfactory index was found by combining both time and acidosis as the integral of the pH change during the period of ischaemia. This was found to have a strong correlation with the histologic changes. There was a less strong correlation between the acidosis during reperfusion and the histologic change. Comparing these results with those for 3 rats subjected to 215 min of ischaemia without reperfusion, it appears that most of the effect of acidosis in aggravating ischaemic injury takes place during the first hour of ischaemia with little further aggravation for longer periods.

Changes in Regional Cerebrovascular Resistance During Partial Cerebral Ischemia in Rats

Secondary changes in regional cerebral blood flow (rCBF) have been observed to occur within hours to days in acute focal cerebral ischemia. Following experimental occlusion of the middle cerebral artery (MCA) the secondary decline in rCBF during the first 4 hours of ischemia appeared to correlate with progressive brain swelling (Hossmann and Schuier, 1980). In subsequent studies the delayed rise in intracortical vascular resistance as the cause of the decrease in rCBF was noted (Shima et al, 1983). It is assumed that the continuing failure of cerebral hemodynamics associated with the delayed rise in regional cerebral vascular resistance (rCVR) can aggravate the ischemia and in this way can contribute to the extension of cerebral infarction.