This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell dea...

Ischemic Cell Death in Brain Neurons

Delayed neuronal death in the gerbil hippocampus following ischemia

We have evaluated the use of 2,3,5-triphenyltetrazolium chloride (TTC) as an histopathologic stain for identification of infarcted rat brain tissue. The middle cerebral artery (MCA) of 35 normal adult rats was occluded surgically. At various times after surgical occlusion, rats were sacrificed and brain slices were obtained and stained with TTC or hematoxolin and eosin (H & E); the size of the area of infarcted tissue stained by each method was quantified. In rats sacrificed 24 hr after occlusion of the MCA, the size of the area of infarction was 21 +/- 2% of the coronal section for TTC, and 21 +/- 2% for H & E (mean +/- S.D., N = 13). The size of areas of infarction determined by either staining method was not significantly different in area by the paired test, and a significant correlation between sizes determined by each method was found by linear regression analysis (r = 0.91, slope = 0.89, and the y intercept = 4.4%). Staining with TTC is a rapid, convenient, inexpensive, and reliable method for the detection and quantification of cerebral infarction in rats 24 hr after the onset of ischemia.

Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.

The plethora of failed clinical trials with neuroprotective drugs for acute ischemic stroke have raised justifiable concerns about how best to proceed for the future development of such interventions. Preclinical testing of neuroprotective drugs is an important aspect of assessing their therapeutic potential, but guidelines concerning how to perform preclinical development of purported neuroprotective drugs for acute ischemic stroke are lacking. This conference of academicians and industry representatives was convened to suggest such guidelines for the preclinical evaluation of neuroprotective drugs and to recommend to potential clinical investigators the data they should review to reassure themselves that a particular neuroprotective drug has a reasonable chance to succeed in an appropriately designed clinical trial. Without rigorous, robust, and detailed preclinical evaluation, it is unlikely that novel neuroprotective drugs will prove to be effective when tested in large, time-consuming, and expensive clinical trials. Additionally, similar recommendations are provided for drugs with the potential to enhance recovery after acute ischemic stroke, a burgeoning new field with great potential but little currently available data. The suggestions contained in this document are meant to serve as overall guidelines that must be adapted to the individual characteristics related to particular drugs and their preclinical and clinical development needs.

Recommendations for standards regarding preclinical neuroprotective and restorative drug development

We have used a photochemical reaction in vivo to induce reproducible thrombosis leading to cerebral infarction in rats. After the intravenous injection of rose bengal, a potent photosensitizing dye, an ischemic lesion was formed by irradiating the left parietal convexity of the exposed skull for 20 minutes with green light (560 nm) from a filtered xenon arc lamp. Animals were allowed to survive from 30 minutes to 15 days after irradiation. Early microscopic alterations within the irradiated zone included the formation of thrombotic plugs and adjacent red blood cell stasis within pial and parenchymal vessels. Scanning electron microscopy revealed frequent platelet aggregates adhering to the vascular endothelium, often resulting in vascular occlusion. Carbon-black brain perfusion demonstrated that occlusion of vascular channels progressed after irradiation and was complete within 4 hours. Histopathological examination at 1, 5, and 15 days revealed that the associated infarct evolved reproducibly through several characteristic stages, including a phase of massive macrophage infiltration. Although cerebral infarction in this model is initiated by thrombosis of small blood vessels, the fact that the main pathological features of stroke are consistently reproduced should permit its use in assessing treatment regimens. Further, the capability of producing infarction in preselected cortical regions may facilitate the study of behavioral, functional, and structural consequences of acute and chronic stroke.

Induction of reproducible brain infarction by photochemically initiated thrombosis

Occlusion of a middle cerebral artery is performed with a surgical clip which is placed through a transorbital approach. This results in a reproducible cerebral lesion (infarction) which was analyzed sequentially in a group of primates at intervals ranging from 2 1/2 hours to 16 days. The earliest cytological alterations, involving neurons, neuropil and astrocytes, can be demonstrated convincingly 2 1/2 hours after the arterial occlusion in paraffin-embedded sections, stained with hematoxylin and eosin. A detailed description of the topographic-cytologic abnormalities and the sequence of inflammatory changes elicited by this form of cerebral ischemia, as well as a delineation of the beginning of the post-ischemic healing processes are provided. A three-zone separation of the histological features is suggested. In addition, we illustrate the cellular alterations in the hemisphere opposite to the arterial occlusion and discuss the axonal changes observed in the different zones of the evolving ischemic encephalomalacia. Abnormalities in the microcirculation are also discussed.

Cerebral infarction. Evolution of histopathological changes after occlusion of a middle cerebral artery in primates.

The feline right cerebral hemisphere was subjected to regional (incomplete) ischemia after clipping the middle cerebral artery for 5, 10, 15, 30 or 60 min, respectively. After each ischemic episode, a 10-min recirculation period was allowed, following which the brain was fixed and processed for electron microscopy. The earliest alterations, detected in the cerebral cortex after 15 min, increased in severity with longer ischemic episodes and were distributed multifocally. There was: (a) marked neuronal mitochondrial matrical swelling and progressive condensation of cytoplasm and nucleoplasm; (b) cytoplasmic swelling of astrocytes with preservation of glial mitochondrial volume; (c) capillaries, oligodendrocytes, myelin sheaths and axis cylinders did not change significantly, even after the longest interval studied: 60 min. This type of tissue reaction appears to be common for those forms of cerebral ischemia, in which circulation is either sustained partially (via collateral arteries) or restored after a period of absolute ischemia. Under these conditions, as yet undefined permeability changes in cell membranes lead to pronounced volumetric alterations of cellular compartments. Although no softening is detectable by digital examination, we suggest that such a set of structural abnormalities constitutes encephalomalacia, or the earliest stage of a lesion which is designated infarction, once it reaches irreversibility.

Cellular events during partial cerebral ischemia. I. Electron microscopy of feline cerebral cortex after middle-cerebral-artery occlusion.

A comparison was made on some of the effects that temporary and permanent occlusion of the middle cerebral artery may have on the circulation to the ipsilateral hemisphere in the cat. Reperfusibility of the corresponding capillary bed was unimpaired for the initial six hours after occluding the vessel, as demonstrated by in vivo intravenous injection of carbon black. Short-term occlusion of the middle cerebral artery, followed by re-opening of the vessel, resulted in either (a) no demonstrable parenchymal lesions (i.e., areas of softening), (b) smaller lesions, or (c) infarctions that were either hemorrhagic or confined to the subcortical white matter of the ipsilateral hemisphere.

Temporary regional cerebral ischemia in the cat. A model of hemorrhagic and subcortical infarction.

Light and electron microscopic features of a cerebellar hemangioblastoma were evaluated with special emphasis on the matter of the origin of interstitial (stroma) cells. Many of the capillary components were of embryonal type, and the stroma cells shared several morphological features with them. It was concluded that the stroma cells originate from vasoformative elements (endothelium and pericytes) and that the endothelial cells are active components of the neoplasm. No convincing morphological counterpart for the erythropoietic activity of this neoplasm was uncovered. Structures identical to ciliary rootlets were identified in many endothelial cells.

https://acsjournals.onlinelibrary.wiley.com/doi/pdf/10.1002/1097-0142%28197306%2931%3A6%3C1528%3A%3AAID-CNCR2820310634%3E3.0.CO%3B2-R

Cerebellar hemangioblastoma: histogenesis of stroma cells.

Permanent, complete global cerebral ischemia was induced in cats by filling the cardiovascular system with a plasma substitute (37° C). At variable intervals and up to 120 min thereafter, these feline brains were perfused with aldehydes and processed for electron microscopy. The resulting cellular alterations were homogeneous and uniform throughout the entire brain; they included early chromatin clumping, gradually increasing electron lucency of the cell sap, distention of endoplasmic reticulum and Golgi cisternae, transient mitochondrial condensation followed by swelling and appearance of flocculent densities, and dispersion of ribosomal rosettes. The marked contrast between the structural alterations in permanent, complete ischemia and incomplete cerebral ischemia, suggest differences in their pathogenesis. A basic determinant factor of the structural changes appears to be the volume of flow (serum, plasma, other) which is available at the time of the injury. This analysis of global cerebral ischemia provides some insight on the nature of cellular changes occurring shortly after somatic death.

Yoshinari Kamijyo

Papers

Cerebral infarction. Evolution of histopathological changes after occlusion of a middle cerebral artery in primates.

Cellular events during partial cerebral ischemia. I. Electron microscopy of feline cerebral cortex after middle-cerebral-artery occlusion.

Temporary regional cerebral ischemia in the cat. A model of hemorrhagic and subcortical infarction.

Cerebellar hemangioblastoma: histogenesis of stroma cells.

The ultrastructure of "brain death". II. Electron microscopy of feline cortex after complete ischemia