Electron microscopy of isolated dog brain

EXPERIMENTAL

NEUROJBGY

Electron

111-119

(1968)

Microscopy

of

20,

Isolated

Dog

Brain

JOSEPH C. LEE, MORRIS B. GLOVER, DAVID D. GILBOE, AND LOUIS BAKAY r

Department of Anatomy University of New and DizGon of

Hospitals,

and Division of Neurosurgery, State York at Buffalo, New York 14214, Neurological Surgery, University Madison, Wisconsin53706

Received

September 2, 1967

The ultrastructure of the isolated and perfused dog brain within the intact cranium appeared normal 1 hr after isolation. There was a slight increase of the vascular permeability in 2 hrs and, in addition, swelling of mitochondria in 3 hrs and 20 min. In each case, neither dilation of astrocytic processes nor enlargement of the intercellular space was observed. The enhanced permeability was indicated by changes in the capillary endothelial cells and the basement membrane.The mitochondrialabnormalitiesoccurred only in certain nerve cells. Introduction

In experimental neurology, the structural and functional complexity of the central nervous system (CNS) is known to constitute, only one aspect of difficulty involved with interpretation of pathological phenomena; another aspect is the continuous interplay between the intra- and extracranial factors. Considerable effort has been made to surmount the latter difficulty by eliminating as many extrinsic factors as possible and yet to preserve the viability of the brain. With the goal of attaining a controllable experimental model of the brain, Heymans and Kochmann (12) devised a technic of isolating the head of the cat, dog and rabbit in 1904. The essence of such a preparation, which has since then been refined and modified (3, 6, 10, 11, 18, 20) is the isolation of blood circulation to and from the head. In leaving the major nerve connections to the body intact, this preparation did not entirely eliminate the possibility of humoral effects and other extracranial influences. White et al. (21, 22) attempted 1 This investigation was supported in part by research grants NB03754 and NBOS%l from the National Institute of Neurological Diseases and Blindness, from the National Foundation for Neuromuscular Diseases, Inc., and R195-66 from the United Cerebral Palsy Research and Education Foundation, Inc. Reprint requests to the Buffalo General Hospital, Buffalo, N.Y. 14203. 111

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to remedy this drawback by developing a procedure that totally isolated the monkey brain from the cranium. They are able to preserve the brain viability by extracorporeal circulation of compatible donor blood. Although the brain in such an extreme situation shows electrocorticai activity for many hours, the increased vascular permeability and edematous changes, which inevitably occur after mere exposure of a small area of the cerebral cortex (1, 19) might be present. Gilboe et al. (7, 8) worked out a compromise procedure in which all the extracranial structures of the isolated head are removed and the viable brain is preserved inside the intact cranial cavity. Their electroencephalographic and biochemical findings indicate that the brain so prepared remains reasonably normal up to 4 hrs or longer (8, 9). The parameter used for brain viability in all the above-mentioned preparations is almost exclusively the electrocortical activity (7, ‘9, 11, 12, 20-22). Most studies concern the metabolic and physiological conditions of the isolated brain (3, 6-9, 18, 20-22) while its morphological aspect, especially at the ultrastructural level, has been much neglected. A viable isolated brain can serve as a useful experimental model for various neurological investigations only when the biochemical and physiological data are corroborated with morphological findings, and electron-microscopic observations of the isolated and perfused dog brain are desirable. Therefore, the present research was undertaken. Material

and

Methods

Three healthy adult mongrel dogs were used and the technique for the procedure of brain isolation and perfusion described by Gilboe and associates was employed (7, 8). The duration of perfusion to maintain viability of the brain varied from 1 to 3 hrs and 20 min. The values of blood flow, pressure, pH, pCO,, PO?, Na+, K+ and Cl- were recorded; the oxygen and glucose consumption, and urea, lactic and pyruvic acid formation were calculated. Toward the end of perfusion with compatible donor blood, warm 0.1 M phosphate-buffer solution and cold 4% glutaraldehyde in the same buffer were pumped through the brain. Tissue fragments were removed from the frontal cortex and white matter, head of caudate nucleus, thalamus, tuber cinereum, hippocampus, tegmenta of midbrain and medulla, area postrema, and the gray and white matter of the upper spinal cord. The specimens were postfixed in phosphate-buffered 1% osmium tetroxide for embedding in Maraglas. The thin sections were stained with lead citrate prior to electron microscopic examination. The details of tissue preparation have been reported (14).

ISOLATED

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Observations

The gross appearance of the isolated brain after perfusion with glutaraldehyde was firm and bloodless. No cerebral swelling could be detected. Our biochemical and physiological findings were comparable to those already published (9). Light microscopy did not reveal any edematouschanges or other abnormalities in these brains. In comparison with the control specimens, the ultrastructure appeared normal in the brain isolated and perfused for 1 hr. No changes were observed even in the upper cervical cord. When the duration of brain isolation and perfusion lasted for 2 hrs, the morphological sign of enhanced vascular permeability, namely the excess formation of large and small pinocytotic vesicles, was seen in some capillaries, particularly in the caudate nucleus, thalamus, and lower brain stem (Fig. 1). This enhanced permeability resulted only in slight hydration of the basement membrane which was widened with patchy areas of low electron density (Fig. 2). The astrocytic processesin the pericapillary region and neuropil did not swell, nor was there any enlargement of the intercellular space in the gray and white

FIG. 1. Thalamus isolated for 2 hrs. A blood capillary has many cytoplasmic flaps (F) which form large pinocytotic vesicles (V). The pericapillary astrocytic processes (Ap) do not swell, nor do the intercellular spaces enlarge. N = nucleus of endothelial cell ; Bm = basement membrane. x 7,000.

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BAKAY

matter of upper cervical cord isolated for 2 hrs. The basement FIG. 2. White membrane (Bm) of a capillary is widened with patchy areas of low electron density. Note the normal appearance of the intercellular spaces, mitochondria (M), myelinated fibers (Mf) and astrocytic processes (Ap). L = lumen of the capillary. x 12,000.

matter. The mitochondria, all cell types were intact (Figs. l-3).

endoplasmic reticulum, and other organelles of in the isolated brain and upper cervical cord

In the brain whose viability had been maintained for 2 hrs and 20 min, neither swelling of the astrocytic processes nor enlargement of the intercellular space were observed (Fig. 4). The numerical increase of large pinocytotic vesicles and slight hydration of the basement membrane were similar to those described above (Figs. l-3). The outstanding alteration in this case was the swelling of mitochondria. The swollen mitochondria were accompanied by dissolution of cristae mitochondriales (Fig. 5). These mitochondrial changes were frequently observed in some nerve cells of the cerebral cortex, but they were less frequent and less severe in other regions of the CNS (Figs. 4. 6). In spite of the presence of swollen mitochondria, other organelles and nuclei of the affected nerve cells did not show any abnormality.

ISOLATED

BRAIN

11.5

FIG. 3. Gray matter of upper cervical cord isolated for 2 hrs. The widening and the patchy areas of low density of the basement membrane (Bm) are evident. The mitochcmdria of dendrites (D), axons (A), myelinated fibers (Mf) and astrocytic processes (Ap) appear normal. F = cytoplasim flap of endothelial cell. x 12,500. Discussion

Our most interesting observation of the isolated dog brain within an intact cranium is the conspicuous absence of both intracellular hydration in the astrocytic processes and extracellular edema in the intercellular space. The slight hydration resulting from the enhanced pinocytotic activity is limited to the basement membrane which can be detected only by electron microscopy. These negative findings indicate that cerebral edema of conventional definition (1) can be prevented in the isolated brain within the cranial cavity kept viable in an artificial, properly prepared physiological environment up to 3 hrs and 20 min. That the increased vascular permeability gives rise to minimal hydration of the basement membrane (Figs. 2, 3) may imply an effective part played by the basement membrane in the blood-brain barrier (4, 5). It is noteworthy that swollen mitochondria occur only in the brain isolated for longer than 3 hrs. Furthermore, these changes are not observed in all the cell types of the isolated CNS, nor are they present in the nerve cells with the same frequency and severity at different levels of the CNS

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FIG. 4. Caudate nucleus isolated for 3 hrs and 20 min. The neuropil is very compact. No swelling of astrocytic processes (Ap) occurs. Except for the slight swelling of the mitochondria (M) in the terminal axons (T), no other changes are present. Mf = myelinated fiber; S = synapse. x 13,500.

(Figs. 5, 6). However, there was a marked increase in glycogen in the astrocytic processes (Figs. 4-6). The actual mechanism underlying such mitochondrial changes is not known, but two possible causes may be discussed. First, it may be the surgical shock in the subject during preparation that is responsible for the ultrastructural changes of mitochondria. Recently the levels of adenosine triphosphate and creatine phosphate were determined in the isolated dog brain. The reduced levels of these highenergy phosphate compounds in many of the isolated brains indicate the onset of shock during the surgical isolation procedure (15). Studies have shown that synthesis of adenosine triphosphate, within the brain mitochondria, is considerably reduced as a consequenceof shock (17). Damage to the systems involved in normal energy transfer could drastically reduce the amount of energy available, and the mitochondria must require the expenditure of energy to maintain their structural integrity. Secondly, it may be the differences in the reactivities of blood vessels that causesthe difference in mitochondrial changes at various levels of the

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FIG. 5. Occipital cerebral cortex isolatedfor 3 hrs and 20 min. Note the severe swelling of mitochondria (M) with dissolution of cristae in the perikaryon of a nerve cell. No changes are observed in the nucleus (N) and the granular endoplasmic reticulum (Er). The synapses (S) and astrocytic processes (Ap) appear normal. T = terminal axon. x 11,000.

CNS (10, 16, 18). The blood vessels in some regions of the brain may react to epinephrine and norepinephrine, causing ischemia, while those in other areas of the brain do not react as readily. There is evidence that ischemia produces enzymatic and ultrastructural changes of the mitochondria (2, 13), It should be mentioned that perfusion pressure often increases with the duration of brain isolation (9, 20). In the present experimentation, the arterial pressure tended to increase during the first and second hour of perfusion. However, the hematocrit reduction and efficient blood filtration had permitted our perfusion for 3 hrs and 20 min at fairly normal pressures. In conclusion, our electron-microscopic observations are in a generally good agreement with the biochemical and physiological findings that the ultrastructure and viability of the isolated brain within the cranium can be preserved by extracorporeal perfusion with compatible donor blood. The increased blood-brain barrier permeability is minimal. If the causesrespon-

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oblongata close to the area postrema isolated for 3 hrs and 20 FIG. 6. Medulla min. The mitochondria (M) of a nerve cell show slight swelling and partial dissolution of cristae. The granular endoplasmic reticulum (Er), astrocytic processes (Ap), myelin sheath (MS) and synapse (S) appear intact. x 11,000.

sible for mitochondrial changes could be avoided, the isolated brain preparation of Gilboe and associates (8) would become an ideal experimental model. References L., and J. C. LEE. 1965. “Cerebral Edema.” Thomas, Springfield, Illinois. N. H. 1961. The cytochemistry of anoxic and anoxic-ischemic encephalopathy in rats. Am. J. Pathol. 38: 587-593. 3. CHUTE, A. L., and D. H. SMYTH. 1939. Metabolism of the isolated perfused cat’s brain. @art. f. Exgtl. Physiol. 29: 379-394. 4. DEMPSEY, E. W., and G. B. WISLOCKI. 195.5. An electron miscroscopic study of the blood-brain barrier in the rat employing silver nitrate as a vital stain. 1.

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