Materials and Methods

All experiments were conducted according to the guidelines issued by the institutional animal care committee of the Henry Ford Hospital and in compliance with regulations formulated by the US Department of Agriculture.¹³

Twenty-eight adult male Wistar rats (body weight, 270 to 310 g) were used in this study; 24 were experimental and 4 were control subjects. Outbred rats were purchased from Charles River Laboratory (Wilmington, Mass), where they were fed Purina rat chow; on arrival in Detroit, they were fed Agway rat food, identical to the Purina chow except for a 1.3% higher protein content. After 1 day of fasting, anesthesia of the animals was induced with 3% halothane and spontaneous respiration of 1.0% to 2.0% halothane in a 2:1 N₂O-O₂ mixture by use of a face mask. A PE-50 catheter in the femoral artery served to monitor arterial blood pressure and to sample blood for analysis of blood gases, glucose, and hemoglobin concentration. Body core temperature of each rat was kept constant (37°C) with a recirculating pad and K module and monitored via an intrarectal type-T thermocouple.

The surgical procedure to occlude a large intracranial artery followed the method described by Zea-Longa et al.¹⁴ Under the operating microscope, the right CCA was exposed through a midline incision. The ECA and the occipital arteries were then ligated with a 5-0 silk suture. The ICA was isolated from the adjacent vagus nerve. Further dissection identified the origin of the pterygopalatine artery, at which point a silk suture was tied loosely around the mobilized ECA, and a microvascular clip was placed across the CCA. An 18- to 19-mm segment of 4-0 nylon monofilament, its tip rounded by heating, was introduced into the ECA at the branching point of the occipital artery. The silk suture placed around the ECA was tightened around the intraluminal nylon monofilament to prevent bleeding, and the clip on the CCA was removed. The nylon monofilament was gently advanced from the ECA into the ICA lumen; the skin incision was closed.

Experiments were terminated at predetermined time intervals: 0.5, 1, 2, 3, 4, 6, 12, and 24 hours after the occlusion of the MCA. There were 3 rats in each experimental subgroup. The controls included in the study were rats subjected to all the same experimental procedures as those described above except that the nylon monofilament was withdrawn within less than 1 minute. Survival times for these control animals were 1, 2, 4, and 24 hours for each animal.

For fixation, under general anesthesia with intraperitoneal ketamine (44 to 80 mg/kg) and xylazine (13 mg/kg), animals were transcardially perfused with either 4% paraformaldehyde in 0.1 mmol/L phosphate buffer for routine histological, histochemical, and immunohistochemical studies or with 3% glutaraldehyde in 0.1 mmol/L phosphate buffer for electron microscopy. Brains fixed with 4% paraformaldehyde were allowed to fix overnight, removed from the skull, and immersed again in paraformaldehyde at 4°C overnight. Next, each brain was cut into five coronal slices of 2-mm thickness each. These were labeled A (frontal) through E (occipital). Histology sections (approximately 6 μm thick) were obtained from paraffin blocks and stained with H&E, LFB-PAS, and Bielschowsky's silver impregnation.

One of the coronal brain slices (level C, corresponding to the anterior commissure) from the group fixed by perfusion with glutaraldehyde was immersed in 3% glutaraldehyde and processed for electron microscopy. Each coronal brain slice was further trimmed into four to five sample pieces (each one measuring 1.0×1.0×2.0 mm). These samples were postfixed in 1% osmium tetroxide for 3 hours, dehydrated through graded ethanols, and embedded in Araldite. Semithin sections (≈1 μm thick) were stained with toluidine blue, and ultrathin sections of the areas of interest, stained with uranyl acetate and lead citrate, were examined with a Philips 300 electron microscope.

For the study of the subcortical white matter section D of the brain (at the level of the splenium), where the subcortical white matter of rat brain has its maximum volume, was selected. On LFB-PAS–stained slides, we selected four nonoverlapping fields in the white matter of the hemisphere ipsilateral to the occluded MCA. In each of these fields, at a magnification of ×150, we completed repetitive measurements of the nuclear diameter of oligodendrocytes and the area in the white matter occupied by vacuoles. These measurements were made on images collected with a charged-coupled-device video-imaging microscope system using computerized cytomorphometric analysis (IMAGIST-2 Image Analysis System–PGT). The same measurements were repeated in four nonoverlapping homotopic fields of the contralateral (nonischemic) hemisphere of each animal and in corresponding fields of both hemispheres in the control animals.

The nuclear diameter of the oligodendrocytes was used as an indicator of the cytoplasmic diameter. The evaluation of the cytoplasmatic volume is technically arduous, but the swelling of the cell nucleus is proportional to that of the cytoplasm, since the nuclear membrane reacts to ischemia in a way similar to the plasma membrane.

Oligodendrocytes were identified at the light microscope level according to conventional criteria as small cells (≤10 μm in diameter) with a round or oval hyperchromatic nucleus and no visible cytoplasm. Astrocytes were identified as cells bigger than oligodendrocytes having a vesicular, brighter, ovoid naked nucleus. The image-analysis program automatically discerned the different brightness of oligodendrocyte and astrocyte nuclei. Small cells with a dark, elongated, and irregularly shaped nucleus were identified as microglia; the morphology of the nucleus allowed for their distinction from oligodendrocytes. Moreover, these cells are few in the white matter,¹⁵ and their involuntary inclusion would not significantly affect the statistical evaluation of oligodendrocyte nuclear diameter.

Results

Data on arterial blood pressure, blood glucose, and blood gases have been published before.² Sham-operated animals did not show any obvious change in the white matter. Changes in the white matter involved oligodendrocytes, astrocytes, and nerve fibers in the hemisphere ipsilateral to the MCA occlusion.

Swelling was the first detectable feature of injured oligodendrocytes. Swollen oligodendrocytes showed a pinkish cytoplasm with H&E staining, a distended nucleus with clumped chromatin in a watery background, and an expanded clear cytoplasm compartment on electron microscopy (Fig 1⇓). These changes were detected in a small number of cells as early as 30 minutes after the occlusion of the MCA, and after 3 hours most cells were swollen. The average diameter of oligodendrocytes as evaluated by the image-analysis system increased progressively in comparison with the contralateral side and with sham controls (Table 1⇓, Fig 2⇓).

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Figure 1.

Top, Normal oligodendrocyte; Wistar rat subjected to sham operation as described in the text (original magnification ×8000). Bottom, Swollen oligodendrocytes; Wistar rat with MCA occlusion 1 hour before death (×2500).

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Figure 2.

Changes in oligodendrocyte nuclear diameters in rat brain after MCA occlusion.

Pyknotic oligodendrocytes, characterized by a distended cytoplasm with a small and hyperchromatic nucleus, emerged during the third hour and became more numerous with time (Fig 3⇓). At 12 and 24 hours the size and number of pyknotic nuclei decreased, overlapping with the appearance of necrotic oligodendrocytes, which were characterized by the presence of bizarre nuclear shapes and fragmentation of chromatin (Table 1⇑, Fig 3⇑). After 24 hours of ischemia, few cells recognizable as oligodendrocytes were detectable in the more severely injured areas, and mostly necrotic cells and swollen astrocytes were present in the center of the lesion. A moderate degree of oligodendroglial nuclear swelling was observed at 3 and 4 hours after ischemia in the contralateral nonischemic hemisphere (Table 1⇑, Fig 2⇑). This contralateral swelling decreased at 6 hours and returned to baseline values at 12 and 24 hours. Pyknotic or necrotic oligodendrocytes never developed in the contralateral hemisphere.

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Figure 3.

Top, Pyknotic oligodendrocyte, swollen axons (asterisks) showing cytoskeletal dissolution, and swollen astrocyte processes (stars); Wistar rat with MCA occlusion 4 hours before death (original magnification ×5000). Bottom, Example of breakdown of plasma and nuclear membranes in oligodendrocytes noted 6 hours after MCA occlusion. Segmental axonal swelling (asterisk) is also noted (×4000).

Astrocyte swelling was readily detectable at 30 minutes after MCA occlusion. The hydropic nuclear swelling provided the cells with an appearance similar to that of Alzheimer II astrocytes. After 4 hours, some cells with pyknotic nuclei were injured astrocytes identified by intracytoplasmic intermediate filaments. At 12 and 24 hours, most viable cells appeared to be astrocytes.

Contemporaneously with the changes in the oligodendrocytes, myelin sheaths in the subcortical white matter lost LFB-PAS stainability. This was associated with the presence of small, empty spaces (vacuoles) separating myelin sheaths (Fig 4⇓). Thirty minutes after the occlusion of the MCA, this white matter vacuolation was easily measured (Table 2⇓, Fig 5⇓). The area of white matter covered by vacuoles increased progressively until 3 hours. The vesicular spaces became the predominant histological feature at 12 and 24 hours. At this time, the more severely injured area in the white matter consisted of vacuoles or cavities alternating with interspersed scant pyknotic nuclei and a rare identifiable cell or neuronal process. In the contralateral hemisphere, isolated vacuolation foci were observed after 3 hours of ischemia.

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Figure 4.

Top, Vacuolated cerebral white matter and pyknotic oligodendrocyte nuclei as seen 3 hours after MCA occlusion (H&E; original magnification ×80). Bottom, Vacuoles in the ischemic white matter correspond to swollen astrocyte processes (stars) and distended myelinated axons (asterisk) as seen 6 hours after MCA occlusion. Oligodendrocyte also shows advanced degenerative changes (×2500). Inset shows the structure of the residual axoplasm (×6200).

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Figure 5.

Changes in area of vacuolation in both hemispheres after unilateral MCA occlusion as a function of time.

The vacuoles observed and quantified by light microscopy (Fig 4⇑, top) corresponded to three ultrastructural alterations. Some represented massively swollen (up to 10 μm in diameter) astrocyte processes, recognizable by their membrane-bound, rounded, irregular outlines and electron-lucent, organelle-free contents (Fig 4⇑, bottom). An additional type of vacuole, visible after 6 hours, resulted from the separation of the myelin sheath from the axolemma; in some areas these spaces expanded into large vacuoles (8 to 10 μm in diameter) surrounded by a thinned but compact myelin sheath (Fig 6⇓, bottom). Changes in nerve fibers, clearly apparent 3 hours after the arterial occlusion, were the third element responsible for the vesicular appearance of the white matter. The vacuoles observed in the nerve fibers corresponded to axons that appeared as irregular, segmentally thickened, twisted profiles with Bielschowsky's stain (Fig 6⇓, top) and ultrastructurally as segmentally distended axons (Fig 3⇑, bottom). After 1 hour axonal compartments expanded, and after 2 hours neither microtubules nor neurofilaments could be identified (Figs 3, top, and 4⇑⇑, bottom, inset). Some distended axons measured up to 8 to 10 μm in diameter.

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Figure 6.

Top, Multiple segmental swellings of myelinated axons are seen in the cerebral white matter 12 hours after MCA occlusion (Bielschowsky's silver impregnation, original magnification ×132). Bottom, At this time (12 hours after MCA occlusion) vacuoles are made up in part of enlarged periaxonal-intramyelinic spaces (asterisk). Axonal contents appear intact in these nerve fibers (stars) (×3150). Inset shows the space between the axon and the adjacent myelin sheath (×4000).

Discussion

In this time-dependent study of cerebral white matter pathology induced by MCA occlusion of up to 24-hour duration, we document widespread injury to oligodendrocytes that precedes pannecrosis by several hours. Vacuolar leukoencephalopathy secondary to axonal and astrocyte swelling are concomitant features of this ischemic lesion.

Despite the common belief that white matter is resistant to ischemia, oligodendrocyte swelling occurred as early as 30 minutes after MCA occlusion, and pyknosis of these cells was widespread after 6 hours. The oligodendroglial injury involves a large proportion of these cells at a time when neuronal damage is evolving and before infarction (pannecrosis) develops, suggesting that a number of glial cells may succumb to the effects of ischemia before morphological expressions of neuronal death become apparent. In most white matter specimens evaluated in this study, the adjacent cerebral cortex and the caudoputamen showed necrosis of only selected isolated neurons. Under identical experimental conditions, the first significant number of necrotic (or eosinophilic) neurons appears after 6 hours in the basal ganglia; however, as late as 24 hours after MCA occlusion only 40% of the cortical neurons are necrotic, and pannecrosis in the preoptic area does not appear until 72 hours after MCA occlusion.^{2 3 16}

To our knowledge, selective oligodendrocyte damage has not been reported in previous experiments of hypoxic-ischemic injury to the cerebral white matter. Increased numbers of oligodendrocytes in the anterior limb of the internal capsule were seen in brains of mice made hypoxic for 2 days,¹⁷ a study in which identification of oligodendrocytes was based on the evaluation of light microscopy samples only. Gerbil brains chronically hypoperfused by the bilateral application of clips to the carotid arteries developed rarefaction of the white matter myelin,¹⁸ but oligodendrocytes were not mentioned in that study; Burger et al¹⁹ reported lesions confined to the centrum semiovale in canine brains made hypoxic and ischemic, but these authors did not provide details about cellular alterations, glial or otherwise.

The remarkable susceptibility of the rat cerebral oligodendrocyte to the type of ischemia described in this study is consistent with the results of in vitro studies showing selective vulnerability of oligodendrocytes and axons to the alterations in intracellular Ca²⁺ concentration induced by anoxia.^{10 21} In addition, altering intracellular Ca²⁺ levels through the local injection of a Ca²⁺ ionophore in the rat spinal cord leads to early oligodendrocyte injury followed by demyelination,²² and Ca²⁺ ionophores have proved lethal to cultured rat oligodendrocytes at concentrations that are not harmful to astrocytes.²³

The nerve fiber injury described here, which was manifested by axonal swelling with dissolution of the cytoskeleton and periaxonal intramyelinic edema, is most likely secondary to the effects of ischemia on the nerve fibers. Selective injury to the spinal white matter was induced in rats after ligating the thoracic aorta and the subclavian arteries for 10 to 12 minutes.²⁴ Furthermore, axonal and glial changes comparable to those we observed were the outcome of three separate experimental situations in which the white matter abnormalities were attributed to hypoxia/ischemia. Largely dilated axons and fragmented and disrupted cell membranes were noted in rat brains exposed to cyanide.^{9 25} Cats exposed to an atmosphere composed of 0.3% CO/air mixture developed segmentally swollen “empty” axons, periaxonal myelin vacuoles, and swollen or necrotic oligodendrocytes.²⁶ The degree and the extent of white matter damage in the feline experiment correlated better with the decrease in blood pressure than with the duration of CO exposure or with the serum levels of carboxyhemoglobin.²⁷ In the third experiment, in vitro axonal and myelinic changes similar to those we document were observed in isolated optic nerves exposed to a 95% N₂ and 5% CO₂ atmosphere.^{21 28}

Collectively, these observations support the hypothesis that the severe damage to nerve fibers that occurs in the white matter after MCA occlusion is the direct effect of hypoxic-ischemic injury and is probably independent of the injury to the neuronal perikarya. Moreover, Wallerian degeneration is a phenomenon that in the central nervous system occurs only several weeks after the neuronal perikarya are destroyed.²⁹

Changes in the cerebral white matter are detected with increasing frequency by neuroimaging methods, particularly in persons aged 60 years or more.¹² These changes, for which the descriptive term “leukoaraiosis” has been proposed,³⁰ are more frequent and severe in older, cognitively impaired subjects,³¹ in whom they seemingly represent the main abnormal finding. Analysis of the existing epidemiological data suggests that cerebral white matter changes may play a role in the development of cognitive disorders among the elderly.³² The etiology and pathogenesis of these white matter alterations are poorly understood, but the most common view is that these changes have an otherwise undefined vascular or ischemic origin. In particular, white matter changes have been presumptively attributed to ischemia of moderate severity,¹¹ and decreased numbers of oligodendrocytes are believed to be part of the leukoencephalopathy.³³ Vague terms such as “pallor” and “rarefaction,” commonly used to describe these alterations of the cerebral white matter, reflect our deficient understanding of their pathogenesis.

The similarity of the changes we document to those attributed to cyanide^{9 25} and carbon monoxide toxicity¹⁰ and to selected features of human leukoencephalopathy^{33 34} suggests that some forms of this condition may be causally associated with ischemia. Permanent occlusion of a large cerebral artery is probably not the primary mechanism leading to this type of brain lesion, but our observations in this experimental model represent the first necessary step in elucidating the effects of ischemia on white matter. We suggest that modifying the mechanism by which brain ischemia is induced could result in the development of relevant models on which we can test hypotheses that explain human subcortical leukoencephalopathy. Future studies of ischemic leukoencephalopathy may be based on transient repeated episodes of ischemia in which the severity of blood flow changes during each episode are of moderate severity.

Selected Abbreviations and Acronyms