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Abstract

The thalamus has been shown to undergo secondary degeneration after cerebrocortical ischemia. However, little is known about the time course of the retrograde thalamic degeneration. The present study was designed to investigate time-dependent changes in the morphology, protein synthesis and calcium metabolism of thalamic neurons in middle cerebral artery (MCA)-occluded spontaneously hypertensive stroke-prone rats that showed primary focal ischemia in the temporoparietal cortex after permanent occlusion of the left distal MCA. In the histologic study by light and electron microscopy, swelling of the nucleus and shrinkage of the perikarya were seen in some neurons of the ventroposterior (VP) thalamic nucleus on the lesioned side at 5 days after ischemia. At the same time, the incorporation of radiolabeled leucine in VP thalamic neurons began to decrease significantly with concomitant a decrease in the number of polyribosomes in the neurons. Conspicuous ⁴⁵Ca accumulation was noted at 3 days after ischemia and persisted up to 1 month in the VP thalamic nucleus on the lesioned side. These findings suggest that the secondary thalamic degeneration after cortical infarction starts with disruption of calcium homeostasis in situ at the third day after MCA occlusion, followed by a decrease in polyribosomes but not by disaggregation of polyribosomes as seen in hippocampal CA1 neurons subjected to transient forebrain ischemia.

Keywords

Focal cerebral ischemia Stroke-prone spontaneous hypertensive rat Thalamic degeneration Protein synthesis Calcium homeostasis Autoradiography

Focal cerebral ischemia is known to cause irreversible neuronal degeneration in brain areas distant from the primary ischemic lesion without any reduction of regional CBF (rCBF) in the areas (Barron et al., 1973; Kataoka et al., 1989; Fujie et al., 1990; Hanyu et al., 1991; Tokuno et al., 1992). For example, permanent occlusion of the unilateral middle cerebral artery (MCA) in rats yields not only infarction in the ipsilateral cerebral cortex and caudoputamen but also secondary degeneration in the ipsilateral thalamus and substantia nigra (Tamura et al., 1981b). Because the cerebral cortex and caudoputamen receive extensive input from the thalamus and substantia nigra, respectively (Iizuka et al., 1989), the secondary neuronal damage as seen in MCA-occluded rats may involve retrograde degenerative processes (Iizuka et al., 1990; O'Gorman and Sidman, 1985a; O'Gorman, 1985b). Thalamic atrophy has also been observed clinically in patients with cerebral infarction in the territory supplied by the MCA, and this atrophy may lead to sustained dementia (Tamura et al., 1991). Thus, protection against ischemia-induced thalamic degeneration, if successfully conducted, would be of clinical value.

The morphologic features of neuronal degeneration in brain areas that are remote from but connected synaptically with the primary infarction site have been studied mainly by conventional light and electron microscopy, and it is indicated that the retrograde degeneration in the areas distant from the primary ischemic lesion is slowly progressive. However, little is known about the time course of the retrograde neuronal degeneration associated with cerebral infarction or about metabolic changes in the sites undergoing retrograde degeneration.

Spontaneously hypertensive stroke-prone (SH-SP) rats with permanent occlusion of the MCA above the rhinal fissure and distal to the striate branches invariably exhibit a certain cortical infarct and subsequent thalamic degeneration (Coyle et al., 1983; Okuyama et al., 1991; Kumon et al., 1996), and they thus appear to be an appropriate subject for the study of secondary neuronal damage in the thalamus. The present study was designed (1) to investigate the time course of thalamic degeneration in SH-SP rats with MCA occlusion by light and electron microscopy, (2) to examine the ischemia-induced changes in the protein synthesis within the thalamus by [¹⁴C]-leucine autoradiography, and (3) to detect the calcium accumulation in the thalamus of MCA-occluded rats by ⁴⁵Ca autoradiography. The results of these experiments should shed light on the starting time of the treatment of secondary thalamic degeneration.

MATERIALS AND METHODS

Animal model

Male SH-SP rats weighing 280 to 320 g were subjected to focal cerebral ischemia with modifications of the method of Brint (1988). Tail systolic pressure in each conscious animal was measured just before MCA occlusion with a rat tail manometer-tachometer system (KN-21.0; Natsume, Tokyo, Japan). During the MCA occlusion procedure, animals were anesthetized with 1.5% halothane in a 2:1 mixture of nitrous oxide and oxygen. The rectal temperature was monitored by a digital thermometer and maintained at 37.0±0.5°C by a heating lamp. A small scalp incision was made at the midpoint between the left outer canthus and the pinna. The temporal muscle was separated and retracted to expose the zygoma and the squamosal bone. With a dental drill, a burr hole was made 1 mm rostral to the anterior junction of the zygoma and the squamosal bone, and the dura was carefully incised. The left MCA was exposed and coagulated at the point distal to the perforating arteries with a bipolar coagulator, and cut to ensure permanent occlusion.

Light microscopy

Rats were anesthetized with pentobarbital and perfused transcardially with 10% formalin in 0.1 mol/L phosphate-buffered saline (PBS) (pH 7.4) at 12 hours; 1, 3, and 5 days; 1 and 2 weeks; and 1, 2, and 3 months after MCA occlusion (four animals each). The brain was removed and embedded in paraffin, and serial coronal sections 2-μm thick were cut. The specimens were stained with hematoxylin and eosin or with 0.1% cresyl violet.

The area of the thalamus in sections at the level 3.6 mm posterior to bregma was measured by a planimeter, and the ratio of the thalamic area on the MCA-occluded side to that on the contralateral side (left/right) was calculated. The number of cells in the ventroposterior (VP) thalamic nucleus was evaluated by measuring the size of each cell with a computerized image processing system (Nexus 6400 System; Kashiwagi, Tokyo, Japan). A square of 0.33 times 0.3 mm (510 times 478 pixels) was designated as a region of interest on the video image. The size of each cell within the square was measured and expressed by its pixel size. Cell number and size were obtained from five regions of interest. Cells larger than 250 pixels (20 μm in diameter) were tentatively regarded as the neuron-rich group, and those smaller than 150 pixels (12 μm in diameter), except for endothelial cells and pyknotic neurons, were regarded as the glia-rich group. Although the cells larger than 250 pixels were considered to be mostly neurons, one could not exclude the possibility that the smaller cells included a few neurons. Therefore, we defined the cells larger than 250 pixels as the neuron-rich group. Although it was not always easy to distinguish between glial cells and degenerating pyknotic neurons, the majority of the pyknotic neurons were as large as 150 to 250 pixels (12 to 20 μm in diameter) and thus could be distinguished from morphologically intact neurons. Moreover, glial cells markedly outnumbered endothelial cells and pyknotic neurons in the rat thalamus. Thus, although pyknotic neurons and endothelial cells were included in the glial-rich group, the overestimation of the glial cell number appeared to be trivial.

Electron microscopy

Rats were anesthetized with pentobarbital and perfused transcardially with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) at 12 hours; 1, 3, and 5 days; 1 week; and 1 month after MCA occlusion (four animals each). After removal of the brain, a tissue block containing the VP thalamic nucleus was dissected, postfixed with 1% osmium tetroxide for 2 hours, dehydrated, and embedded in epoxy resin. Neurons in the VP thalamic nucleus were identified on semi-thin sections stained with 1 % toluidine blue, and then ultrathin sections 70-nm thick were cut on an ultramicrotome. The ultrathin sections were examined with a transmission electron microscope (H-800; Hitachi, Tokyo). The number of polyribosomes (aggregation of more than four ribosomes) per 1 μm² in the basal side of a perikaryon was assessed in 40 randomly chosen neurons of the VP thalamic nucleus by using a micro computer imaging device (MICD) image analyzer (Imaging Research, St. Catherine, Ontario, Canada).

Measurement of rCBF

rCBF was measured by [¹⁴C] iodoantipyrine autoradiography at 1, 3, 6, and 12 hours; 1 and 3 days; 1 week; and 1 month after MCA occlusion (three animals each). The femoral vein was cannulated under pentobarbital anesthesia, and a bolus of 50 μCi/kg of 4-iodo [N-methyl-¹⁴C] antipyrine (specific activity, 67.5 mCi/mmol; New England Nuclear, Boston, MA) was injected intravenously over a 30-second period with a microsyringe (Hamilton, Reno, NV). The animal was decapitated approximately 30 seconds after the start of isotope injection. The brain was promptly removed and frozen with dry-ice-cold isopentane. Coronal sections 20-μm thick were cut with a cryostat and mounted on poly-L-lysine—coated slide glasses. Coronal sections for macroautoradiography were exposed to Kodak NMC-1 film (Eastman Kodak, Rochester, NY) for 2 weeks to detect regional radioactivity in the thalamus and in the cerebral cortex. The mean optical density of five boxed areas (containing 100 pixels in 0.01 mm² each) in the VP thalamic nucleus and in the cerebral cortex on both sides were calculated by using a MICD imaging analyzer and the standard optical densities of coexposed ¹⁴C-Microscales (Amersham, Arlington Heights, IL).

[14C]-leucine autoradiography

Amino acid incorporation in the thalamus was investigated by [¹⁴C]-leucine autoradiography at 12 hours; 1, 3, and 5 days; 1 and 2 weeks; 1 and 2 months after MCA occlusion (four animals each). The femoral vein was cannulated under pentobarbital anesthesia, and a bolus of 150 μCi/kg of L-[¹⁴C]leucine (C₁-labeled; specific activity, 58.0 mCi/mmol; New England Nuclear) was injected intravenously with a Hamilton microsyringe. The animal was decapitated 45 minutes after the injection. The brain was promptly removed and frozen with dry-ice-cold isopentane. Coronal sections of 10- and 20-μm thickness were cut with a cryostat in alternate order, and mounted on poly-L-lysine—coated slide glasses. All sections were incubated with 10% trichloroacetic acid overnight, and rinsed with water for 5 minutes to remove nonincorporated [¹⁴C]-leucine. Twenty-μm sections for macroautoradiography were exposed to Kodak NMC-1 film for 4 weeks to detect regional radioactivity in the thalamus. The mean optical density of five boxed areas (containing 100 pixels in 0.01 mm² each) in the VP thalamic nucleus on both sides were calculated and converted to radioactivity per tissue weight (nCi/g) using the MICD imaging analyzer and the standard optical densities of coexposed ¹⁴C-Microscales (Amersham).

Ten-μm sections were used for microscopic autoradiography after dehydration (Stumpf et al., 1991). NR-M2 (Konica, Tokyo) was applied to the sections which were then exposed for 4 weeks at 4°C. The incorporated [¹⁴C]-leucine, manifested as black deposits, was observed under a light microscope at x400 magnification.

45Ca autoradiography

Calcium accumulation was investigated by ⁴⁵Ca autoradiography at 1 and 12 hours; 1, 3, and 5 days; 1 and 2 weeks; and 1 month after MCA occlusion (four animals each). Animals were anesthetized with pentobarbital and the femoral vein was cannulated. A bolus of 1 mCi/kg of ⁴⁵Ca (Ca-labeled; specific activity, 6.75 Ci/g; ICN Biomedicals Inc, CA, USA) was injected intravenously with a Hamilton microsyringe 6 hours before decapitation. The brain was promptly removed and frozen with dry-ice-cold isopentane. Coronal sections of 10- and 20-μm thickness were cut with a cryostat in alternate order, and mounted on poly-L-lysine—coated slide glasses. Twenty-micrometer sections were exposed to Kodak NMC-1 film for 4 weeks. The mean optical density of five boxed areas (containing 100 pixels in 0.01 mm² each) in the VP thalamic nucleus on both sides were calculated by the MICD imaging analyzer.

Ten-micrometer sections were used for microscopic autoradiography after dehydration. NR-M2 (Konica) was applied to the sections which were then exposed for 4 weeks at 4°C. The accumulated ⁴⁵Ca, manifested as black deposits, was observed under a light microscope at x400 magnification.

Statistical analysis

All values were given as mean±SD. Statistical comparisons among experimental groups were performed by the two-factor analysis of variance, followed by Scheffé's test for multiple comparisons. Statistical comparisons among data in the left and right hemispheres of the same animals were performed by paired t-test.

RESULTS

Light microscopy

In SH-SP rats with occlusion of the left MCA above the rhinal fissure and distal to the striate branches, the infarction site was confined to the left temporoparietal cortex, and the size of infarction was similar in all rats. A significant reduction in the ratio of the ipsilateral thalamic area to the contralateral one was noted 1 month after MCA occlusion, and it was further enhanced 3 months after ischemia (Fig. 1). Among the thalamic nuclei, the left VP thalamic nucleus, which projects nerve fibers to the infarcted area, specifically showed progressive shrinkage.

FIG. 1.

Time-dependent changes in the ratio of the left thalamic area to the right thalamic area after left middle cerebral artery occlusion. There were significant decreases in the ratio at 1 and 3 months after ischemia. Bars represent the SD. *0 day indicates normal control. Significant differences (*P < .05, **P < .01) are noted between ipsilateral and contralateral thalamus (paired t- test).

At high magnification, swelling of the cell nuclei and shrinkage of the neuronal perikarya were observed in the left but not right VP thalamic nucleus at the fifth day after MCA occlusion (Fig. 2A, B). One month after MCA occlusion, many neurons in the left VP thalamic nucleus had dark-stained pyknotic nuclei and shrunken perikarya, and glial cells were increased in number within the thalamic nucleus (Fig. 2C). Cell counting by the computerized image processing system revealed that neurons in the left VP thalamic nucleus were less numerous than those in the right VP thalamic nucleus at the seventh day after MCA occlusion and decreased gradually in number thereafter, and that the left VP thalamic nucleus contained more glial cells than the right one at the fifth day after MCA occlusion and exhibited a time-dependent increase in glial cells (Fig. 3).

FIG. 2.

Photomicrographs of the ventroposterior thalamic nucleus in 1–μm paraffin sections stained with cresyl violet. (A) normal control; (B) 5 days after MCA occlusion; (C) 1 month after MCA occlusion. Note swelling of the cell nucleus and shrinkage of the perikarya in a neuron in Figure 2B. Degenerating neurons with dark-stained pyknotic nuclei (arrow) and many glial cells are seen in the VP thalamic nucleus at 1 month after MCA occlusion (C). Bar = 40 μm.

FIG. 3.

Time-dependent changes in the ratio of the number of ventroposterior (VP) thalamic neurons (closed circles) or glial cells (closed triangles) on the lesioned side to that of VP thalamic neurons or glial cells on the control side. There were significant decreases in the left-to-right ratio of neurons during the period 1 week to 3 months after middle cerebral artery (MCA) occlusion. In contrast, the left-to-right ratio of glial cells began to increase at the fifth day after MCA occlusion. Bars represent the SD. *0 day indicates normal control. Significant differences (*P < .05, **P < .01) are noted between ipsilateral and contralateral VP thalamic nuclei (paired t-test).

Electron microscopy

Electron micrographs of surviving neurons in the left VP thalamic nucleus are shown in Figure 4. Many ribosomes formed rosette-like aggregations, i.e., polyribosomes, in the perikarya of normal neurons (Fig. 4A). Perikaryal polyribosomes began to decrease in number at 5 days after the ischemic insult without showing any disaggregation of individual ribosomes (Fig. 4B, Table 1). At this period, thalamic neurons in the course of secondary degeneration exhibited partial vacuolation of the cytoplasm (Fig. 4B). During the period from 7 days to 1 month after MCA occlusion, the perikaryal polyribosomes gradually decreased in number (Fig. 4C, D, Table 1), and cytoplasmic vacuolation and mitochondrial swelling became conspicuous in a time-dependent manner. Besides the above changes in the cytoplasm, a deep indentation of cell nucleus and cell shrinkage associated with an abnormal high electron density were noted in a number of thalamic neurons at more advanced stage of degeneration (Fig. 5A, B).

FIG. 4.

Electron micrographs of neuronal perikarya in the left ventroposterior thalamic nucleus. (A) normal control; (B) 5 days after middle cerebral artery (MCA) occlusion; (C) 1 week after MCA occlusion; (D) 1 month after MCA occlusion. Vacuolation was seen at the fifth day after MCA occlusion (B; arrow). Note the decrease in the number of polyribosomes and swelling of mitochondria (arrows) at 1 week after MCA occlusion (C), and the further reduction of polyribosomes at 1 month after ischemia (D). Disaggregation of individual ribosomes forming the polyribosomes was not seen throughout the period of investigation. Bar = 1 μm.

FIG. 5.

Electron micrographs showing a deep indentation of the cell nucleus of a thalamic neuron at the fifth day after middle cerebral artery (MCA) occlusion (A) and shrinkage of a nerve cell body with abnormally high electron density at the 1st week after MCA occlusion (B) at more advanced stage of degeneration. Bar = 1 μm

TABLE 1.

Time-dependent changes in polyribosome number in the ventroposterior thalamic nucleus after MCA occlusion in SH-SP rats

n = 4 each	Ipsilateral(left)(number/μm²)	Contralateral(right)(number/μm²)	%*
0 day†	101.6 ± 13.2	102.3 ± 14.9	99.3%
12 hours	82.4 ± 11.3	88.3 ± 9.6	93.3%
1 day	104.4 ± 15.8	90.1 %15.1	115.9%
3 days	77.7 ± 14.5	80.5 ± 10.7	96.6%
5 days	64.8 ± 19.5‡	81.3 ± 19.8	79.7%§
1 week	46.1 ± 9.5∥	71.8 ± 6.0	64.2%¶
1 month	38.7 ± 3.0∥	72.2 ± 15.2	53.6%¶

Values are mean ± SD. Sh-SP, spontaneously hypertensive stroke-prone.

†

0 day indicates normal control.

Mean left-to-right ratio.

Significant difference († P < 0.05, ∥P < 0.01) between the ipsilateral and normal thalamic nuclei (Scheffé's test).

Significant difference (§P < 0.05, ¶P < 0.01) between the ipsilateral and contralateral thalamic nuclei (paired-t test).

[14C] iodoantipyrine autoradiography

Table 2 shows the left-to-right ratio (light/right) of [¹⁴C] iodoantipyrine radioactivity in the cerebral cortex and in the VP thalamic nucleus. [¹⁴C] iodoantipyrine autoradiography showed that there was no significant difference in rCBF between the right and left thalamus until 1 month after the ischemic insult, whereas in the left temporoparietal cortex, rCBF severely decreased during 1 hour to 1 week after MCA occlusion (Table 2).

TABLE 2.

Time-dependent in accumulated [¹⁴C] iodoantipyrine radioactivity in the ventroposterior thalamic nucleus and cerebral cortex after MCA occlusion in SH-SP rats

n = 3 each	VP nucleus(Ipsilateral/contralateral)	Cortex(Ipsilateral/contralateral)
1 hour	1.03 ± 0.22	0.011 ± 0.010^*
3 hours	1.03 ± 0.14	0.025 ± 0.009^*
6 hours	0.96 ± 0.16	0.016 ± 0.007^*
12 hours	1.01 ± 0.14	0.059 ± 0.012^*
1 day	0.97 ± 0.11	0.021 ± 0.007^*
3 days	0.94 ± 0.14	0.121 ± 0.019^*
1 week	1.01 ± 0.09	0.134 ± 0.016^*
1 month	0.86 ± 0.21	—

Values are mean ± SD. SH-SP, spontaneously hypertensive stroke-prone; VP, ventroposterior.

Significant difference (* P < 0.01) between brain areas ipsilateral and contralateral to the ischemic lesion (paired-t test).

L-[14C]-leucine autoradiography

The macroscopic L-[¹⁴C]-leucine autoradiography showed that there was no difference in amino acid incorporation between the right and left thalamus at the first day after the ischemic insult, while the amino acid incorporation was severely suppressed in the infarcted temporoparietal cortex at this time (Fig. 6A, Table 3). At the fifth day after the MCA occlusion, the amino acid incorporation in the ipsilateral VP thalamic nucleus was significantly reduced in comparison with that in the contralateral thalamus (Fig. 6B, Table 3). However, it returned close to the value of the contralateral thalamus during the period 1 to 2 weeks after ischemic insult (Fig. 6C, Table 3). At 1 month after the MCA occlusion, the amino acid incorporation in the VP thalamic nucleus on the infarction side was reduced again (Fig. 6D, Table 3). Such a transient postischemic resumption of amino acid incorporation as seen in the left VP thalamic nucleus was also noted in the infarcted temporoparietal cortex (Fig. 6).

FIG. 6.

L-[¹⁴C]-leucine macroautoradiographs. (A) 1 day; (B) 5 days; (C) 1 week; (D) 1 month after middle cerebral artery (MCA) occlusion. [¹⁴C]-leucine incorporation in the left ventroposterior (VP) thalamic nucleus was impaired at the fifth day after MCA occlusion (B, arrow). However, transient recovery of [¹⁴C]-leucine incorporation was seen in the left VP thalamic nucleus at 1 week after MCA occlusion (C; arrow). [¹⁴C]-leucine incorporation was reduced again at 1 month after ischemia (D, arrow).

L-[¹⁴C]-leucine microscopic autoradiographs are shown in Figure 7. One day after MCA occlusion, deposits representing L-[¹⁴C]-leucine incorporation were seen mainly in and around thalamic neurons (Fig. 7A). They decreased in number at the fifth day after ischemia (Fig. 7B). During the period 1 to 2 weeks after MCA occlusion, the deposits were observed mainly in and around glial cells (Fig. 7C). At 1 month after MCA occlusion, L-[¹⁴C]-leucine incorporation was scarcely observed in neurons or glial cells (Fig. 7D).

FIG. 7.

L-[¹⁴C]-leucine microautoradiographs in the ventroposterior (VP) thalamic nucleus on the lesioned side. (A) 1 day; (B) 5 days; (C) 1 week; (D) 1 month after middle cerebral artery (MCA) occlusion. Black deposits that represented incorporated [¹⁴C]-leucine were recognized mainly in and around neurons at the first day after MCA occlusion (A), and were decreased in number at the fifth day after ischemia (B). Deposits in and around neurons decreased in number and those in and around glial cells increased in number at 1 week after the MCA occlusion (C; arrow). Thereafter, the deposits were decreased in number throughout the VP thalamic nucleus (D). Bar = 40 μm.

TABLE 3.

Time-dependent changes in incorporated [¹⁴C] leucine radioactivity in the ventroposterior thalamic nucleus after MCA occlusion in SH-SP rats

n = 3 each	Ipsilateral(left)(nCi/g)	Contralateral(right)(nCi/g)	%^*
0 day†	175.5 ± 13.1	167.7 ± 14.0	105.5% ± 16.6%
12 hours	152.7 ± 22.1	153.8 ± 28.5	99.3% ± 13.1%
1 day	162.1 ± 12.0	168.6 ± 10.1	96.1% ± 7.7%
3 days	133.2 ± 11.5	155.7 ± 14.5	85.5% ± 10.1%
5 days	127.5 ± 18.6‡	161.1 ± 24.6	79.1% ± 12.4%§
1 week	139.2 ± 47.3	153.8 ± 42.1	90.5% ± 14.7%
2 weeks	147.0 ± 9.8	151.5 ± 14.4	97.0% ± 11.8%
1 month	119.4 ± 14.2^∥	147.0 ± 26.5	81.2% ± 12.9%¶

Values are mean ± SD. SH-SP, spontaneously hypertensive stroke-prone.

†

0 day indicates normal control.

Mean left-to-right ratio.

Significant difference († P < 0.05, ∥ P < 0.01) between the ipsilateral and normal thalamic nuclei (Scheffé's test).

Significant difference (¶P < 0.05, §P < 0.01) between the ipsilateral and contralateral thalamic nuclei (paired-t test).

45Ca autoradiography

The macroscopic ⁴⁵Ca autoradiography showed ⁴⁵Ca accumulation only in the infarcted temporoparietal cortex at 1 hour after MCA occlusion (Fig. 8A, Table 4). ⁴⁵Ca accumulation occurred in the VP thalamic nucleus on the lesioned side by 3 days after MCA occlusion, and extended markedly into the entire temporoparietal cortex (Fig. 8B, Table 4). The accumulation of ⁴⁵Ca in the left VP thalamic nucleus persisted even after an abrupt decrease in ⁴⁵Ca accumulation in the infarcted temporoparietal cortex on postischemic day 14 (Fig. 8C, Table 4).

FIG. 8.

⁴⁵Ca macroautoradiographs. (A) 1 hour; (B) 3 days; (C) 2 weeks after middle cerebral artery (MCA) occlusion. Calcium accumulation was observed at 1 hour after MCA occlusion only in the left (ischemic) cerebral cortex (A). Calcium accumulation was also seen in the VP thalamic nucleus at the third day after ischemia (B; arrow). Thereafter, the calcium accumulation persisted in the VP thalamic nucleus, while it was reduced in the primary infarcted area by 2 weeks after ischemic insult (C).

TABLE 4.

Time-dependent in accumulated [⁴⁵Ca] radioactivity in the ventroposterior thalamic nucleus and cortex after MCA occlusion in SH-SP rats

n =3 each	VP nucleus(Ipsilateral/contralateral)	Cortex(Ipsilateral/contralateral)
1 hour	1.12 ± 0.12	2.31 ± 0.45*
12 hours	1.10 ± 0.09	3.25 ± 0.48*
1 day	1.15 ± 0.16	6.36 ± 0.49*
3 days	2.89 ± 0.24*	6.77 ± 0.43*
5 days	2.65 ± 0.33*	7.25 ± 0.89*
1 week	2.51 ± 0.29*	5.53 ± 0.91*
2 weeks	3.08 ± 0.76*	1.79 ± 0.19*
1 month	2.69 ± 0.48*	1.76 ± 0.18*

Values are mean ± SD. SH-SP, spontaneously hypertensive stroke-prone. VP, ventroposterior.

Significant difference (P < 0.01) between brain areas ipsilateral and contralateral to the ischemic lesion (paired-t test).

The microscopic ⁴⁵Ca autoradiography performed at the third day after MCA occlusion showed that more deposits representing ⁴⁵Ca radioactivity accumulated mainly in and around the neurons of the left and not right VP thalamic nucleus (Fig. 9).

FIG. 9.

⁴⁵Ca microautoradiographs in the bilateral ventroposterior (VP) thalamic nucleus at the third day after middle cerebral artery occlusion. (A) left (lesioned) side; (B) right (control) side. Black deposits that represented accumulated calcium radioactivity were seen mainly in and around neurons of the left VP thalamic nucleus (A), and they were scattered in the right VP thalamic nucleus (B). Bar = 20 μm.

DISCUSSION

In the present study, SH-SP rats with occlusion of the MCA distal to the striate branches were used as a model to evaluate degeneration of the thalamus, which is remote from but connected synaptically with the primary ischemic lesion, because these rats exhibit a uniform and reproducible infarction localized only in the temporoparietal cortex, possibly due to little collateral pial anastomosis. We found that the blood flow of the thalamus was not affected in MCA-occluded SH-SP rats. This enabled us to investigate secondary thalamic degeneration without the effect of blood flow changes on the thalamus. In contrast, the conventional MCA-occlusion model using a different rat strain involves infarction not only in the temporoparietal cortex but also in the caudoputamen (Tamura et al., 1981a). Because both infarcted regions have synaptic connections with the thalamus and the size of the primary infarcted area varies considerably from rat to rat in the conventional MCA-occlusion model, it is difficult to quantitatively analyze the primary ischemic neuronal damage and subsequent thalamic degeneration. Taken together, the results of the present study suggest that our ischemic model using SH-SP rats may be more suitable for the investigation of thalamic degeneration after cortical ischemic damage than is the model previously reported.

Although there are several reports depicting thalamic degeneration associated with cerebral infarction, they refer only to relatively short-term morphologic alterations such as shrinkage of the thalamus 2 weeks after ischemia (Barron et al., 1973) and the development of neuronal abnormalities 1 week after ischemia (Iizuka et al., 1989, 1990; Yamada et al., 1991). In the present study, neuronal changes including cellular swelling and a decrease in polyribosomes in the VP thalamic nucleus on the lesioned side were observed 5 days after the MCA occlusion, and a significant reduction in the thalamic area was noted 1 month later. These morphologic changes are in agreement with the findings of previous studies (Iizuka et al., 1989, 1990; Fujie et al., 1990; Yamada et al., 1991) and reminiscent of the chromatolysis observed in neurons of the dorsal root ganglia after their axon injury (Evans and Gray, 1961; Meller, 1989). However, the VP thalamic neurons undergoing secondary degeneration, unlike the dorsal ganglion neurons subjected to process injuries, did not exhibit any disaggregation of individual ribosomes forming polyribosomes in harmony with a time-dependent decrease in the number of polyribosomes. In other words, chromatolysis did not occur in the thalamic neurons in response to the cerebral infarction in SH-SP rats. This finding is in accord with the results of previous studies using other focal ischemia models (Holmes, 1906; Iizuka et al., 1990).

In this study, however, we did not find nuclear chromatin fragmentation or apoptotic bodies throughout the experimental period; only a deep indentation of cell nucleus and cell shrinkage were noted in thalamic neurons at more advanced stages of degeneration. Although some of the degenerating thalamic neurons are known to exhibit DNA fragmentation as evaluated by deoxyribo-nucleotidyl transferase—mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) (Watanabe et al., 1997), they don't show all ultrastructures characteristic of apoptosis. We speculate that the secondary thalamic degeneration after cereberocortical infarction is a thalamus-specific event different from apoptosis or necrosis.

There have been a few studies showing metabolic or functional changes in the degenerating thalamus after cortical and striate infarction. In these study, reduction of glucose metabolism and suppression of somatosensory-evoked potentials were recognized at the first day after MCA occlusion (Kataoka et al., 1989; Tokuno et al., 1992). However, no report has focused on changes in protein synthesis in the degenerating thalamus after a selective cortical infarct. We examined radiolabeled amino acid incorporation to investigate the ability of protein synthesis in VP thalamic neurons after MCA occlusion, using L-[¹⁴C]-leucine as a tracer. It is adsorbed by the brain tissue at a high ratio (Oldendorf, 1971) and incorporated exclusively into newly synthesized proteins without remaining in the free radioactive form (Ingvar et al., 1985). The possibility of erroneous overestimation of the amino acid incorporation due to an increase in the specific activity of precursor pool must be considered under pathologic conditions when we interpret the data obtained from tracer-dose autoradiography because tracer-dose autoradiography with labeled amino acids cannot determine the specific activity of the precursor pool (Bodsch and Hossmann, 1983; Furuta et al., 1993). Nevertheless, a reduction in focal radioactivity as determined by tracer-dose amino acid autoradiography appears to reflect inhibition of protein synthesis in situ (Bodsch and Hossmann, 1986; Yoshimine et al., 1987).

In the present macroautoradiography, the amino acid incorporation into the VP thalamic nucleus on the lesioned side was reduced at the fifth day after cortical infarction, and it transiently returned close to the level of the control VP thalamic nucleus during 1 to 2 weeks after MCA occlusion. The microautoradiography showed that while deposits representing radioactivity were recognized mainly in and around neurons of the VP thalamic nucleus at the first and fifth day after MCA occlusion, they began to localize to glial cells at the first and second weeks after MCA occlusion. Therefore, the transient resumption of amino acid incorporation as detected in the macroautoradiography during 1 to 2 weeks after MCA occlusion is likely to reflect an increase in [¹⁴C]-leucine incorporation by reactive astrocytes, and amino acid incorporation in the thalamic neurons appears to decrease gradually after MCA occlusion. The suppression of amino acid incorporation in the neurons took place in accordance with the time-dependent decrease in the number of polyribosomes. These findings suggest that protein synthesis in the thalamic neurons decreased gradually after ipsilateral cortical infarction. In the gerbil hippocampal CA1 region, in which delayed neuronal death occurs after transient forebrain ischemia, polyribosomes in the perikarya of CA1 pyramidal neurons are abruptly diaggregated and amino acid incorporation is severely impaired within several hours after ischemic loading (Furuta et al., 1993). However, we observed that in rat VP thalamic neurons in the course of secondary degeneration, protein synthesis was maintained until 3 days after MCA occlusion and it gradually decreased thereafter. The question remains as to whether the decrease in protein synthesis is an epiphenomenon concurrent with the process of cell death or the cause of cell death. Protein synthesis appears to be crucial for cell survival on the basis of the finding that the application of protein synthesis inhibitors induces cell death in various organs (Cidlowski, 1982), but protein synthesis inhibitors prevent delayed neuronal death after transient global ischemia (Shigeno et al., 1990). Perhaps, inhibition of protein synthesis has diverse actions on damaged cells, depending on types, degrees, and stages of cell damage.

Among several methods for the measurement of the intracellular calcium level (Harris et al., 1981; Krnjevic et al., 1986; Deshpande et al., 1987), we selected ⁴⁵Ca autoradiography to evaluate changes in Ca²⁺ in the VP thalamic nucleus because this technique reflects the in vivo distribution of calcium in tissue and tissue fluids at the time of ⁴⁵Ca administration, despite the technique's poor resolution and difficulty in quantification (Nakamura et al., 1993). ⁴⁵Ca accumulated in the cortical infarcted area as early as 1 hour after the MCA occlusion, whereas the VP thalamic nucleus on the lesioned side began to exhibit ⁴⁵Ca accumulation at the third day after MCA occlusion, before the significant decrease in protein synthesis within the brain nucleus. Microautoradiographs showed that the ⁴⁵Ca accumulated mainly in neurons of the VP thalamic nucleus. An overaccumulation of intracellular Ca²⁺ can induce cell death, acting occasionally as a setpoint of programmed cell death (Cohen, 1993). It can also exacerbate neuronal death in cases of cerebral ischemia. When hippocampal CA1 neurons vulnerable to brain ischemia show delayed neuronal death, an overload of intracellular Ca²⁺ is considered to inhibit protein synthesis, leading to cell death. It is plausible that the increase in the intracellular Ca²⁺ concentration before the decrease in protein synthesis in thalamic neurons of MCA-occluded SH-SP rats accelerates the slowly progressive thalamic degeneration. Presumably, the early Ca²⁺ increase in cases of brain ischemia leads to a decrease in polyribosomes in thalamic neurons or to a disaggregation of polyribosomes in hippocampal CA1 neurons, thereby inhibiting protein synthesis in different manners within the thalamic and CA1 neurons. The mechanisms by which Ca²⁺ regulates protein synthesis differentially remain to be elucidated.

Based on the present experimental results, the secondary thalamic degeneration is concluded to be a slowly progressive event after the primary cortical lesion has been established. The possibility of prevention of the thalamic degeneration is apparently greater than that of the prevention of the primary ischemic damage. Indeed, there is a time difference between the beginning of calcium accumulation and the suppression of protein synthesis in the thalamus after the onset of ischemia. It is tempting to speculate that the secondary thalamic degeneration could be prevented if neuroprotective agents are administered before the increase in the Ca²⁺ concentration in the thalamus. This speculation is supported by the finding that thalamic degeneration after cortical infarction is precluded by the intracisternal administration of basic fibroblast growth factor (Yamada et al., 1991) or by continuous infusion of ciliary neurotrophic factor into the lateral ventricle (Kumon et al., 1996) at early periods after ischemia. Further studies are needed to determine whether the prevention of secondary neuronal damage is beneficial together with treatment of the primary ischemic lesion.

Footnotes

Acknowledgments

The authors thank Professor T. Suzuki and Dr. Y. Ohta (Kinki University) for the generous provision of SH-SP rats, and Mr. M. Sudoh, Mr. S. Masuda, and Mr. D. Shimizu for their technical assistance.

Abbreviations used

References

Barron

Means

Larsen

(1973) Ultrastructure of retrograde degeneration in thalamus of rat. 1. Neuronal somata and dendrites. J Neuropathol Exp Neurol 32:218–244

Brint

Jacewicz

Kiessling

Tanabe

Pulsinelli

(1988) Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 8:474–485

Bodsch

Hossmann

(1983) A quantitative regional analysis of amino acids involved in rat brain protein synthesis by high performance liquid chromatography. J Neurochem 40:371–382

Bodsch

Barbier

Oehmichen

Grosse Ophoff

Hossmann

(1986) Recovery of monkey brain after prolonged ischemia. II. Protein synthesis and morphological alteration. J Cereb Blood Flow Metab 6:22–33

Cidlowski

(1982) Glucocorticoids stimulate ribonucleic acid degeneration in isolated rat thymic lymphocytes in vitro. Endocrinology 111:184–190

Cohen

(1993) Apoptosis. Immunol Today 14:126–130

Coyle

Jokelainen

(1983) Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats. Stroke 14:605–611

Deshpande

Siesjö

Wieloch

(1987) Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia. J Cereb Blood Flow Metab 7:89–95

Evans

DHL

Gray

(1961) Changes in the fine structure of ganglion cells during chromatolysis. Proc Anat Soc Great Britain and Ireland 1:71–74

10.

Fujie

Kirino

Tomukai

Iwasawa

Tamura

(1990) Progressive shrinkage of the thalamus following middle cerebral artery occlusion in rats. Stroke 21:1485–1488

11.

Furuta

Ohta

Hatakeyama

Nakamura

Sakaki

(1993) Recovery of protein synthesis in tolerance-induced hippocampal CA1 neurons after transient forebrain ischemia. Acta Neuropathol 86:329–336

12.

Hanyu

Abe

Arai

Iwamoto

Katsunuma

(1991) Effects of cerebral cortical lesions on the ipsilateral thalamus. Brain Nerve 43:255–261

13.

Harris

Symon

Branston

Bayhan

(1981) Changes in extracellular calcium activity in cerebral ischemia. J Cereb Blood Flow Metab 1:203–209

14.

Holmes

(1906) On the relation between loss of function and structural change in focal lesions of the central nervous system, with special reference to secondary degeneration. Brain 29:514–523

15.

Iizuka

Sakatani

Young

(1989) Selective cortical neuronal damage after middle cerebral artery occlusion in rats. Stroke 20:1516–1523

16.

Iizuka

Sakatani

Young

(1990) Neural damage in the rat thalamus after cortical infarcts. Stroke 21:790–794

17.

Ingvar

Maeder

Sokoloff

Smith

(1985) Effects of ageing on local rates of cerebral protein synthesis in Sprague-Dawley rats. Brain 108:155–170

18.

Kataoka

Hayakawa

Yamada

Mushiroi

Kuroda

Mogami

(1989) Neuronal network disturbance after focal ischemia in rats. Stroke 20:1226–1235

19.

Krnjevic

Morris

Ropert

(1986) Changes in free calcium ion concentration recorded inside hippocampal pyramidal cells in situ. Brain Res 374:1–11

20.

Kumon

Sakaki

Watanabe

Nakano

Ohta

Matsuda

Yoshimura

Sakanaka

(1996) Ciliary neurotrophic factor attenuates spatial cognition impairment, cortical infarction and thalamic degeneration in spontaneously hypertensive rats with focal cerebral ischemia. Neurosci Lett 206:141–144

21.

Meller

(1989) Chromatolysis of dorsal root ganglion cells studied by cryofixation. Cell Tissue Res 256:283–292

22.

Nakamura

Hatakeyama

Furuta

Sakaki

(1993) The role of early Ca²⁺ influx in the pathogenesis of delayed neuronal death after brief forebrain ischemia in gerbils. Brain Res 613:181–192

23.

Okuyama

Shimamura

Karasawa

Kawashima

Araki

Kimura

Otomo

Aihara

(1991) Protective effects of minaprine in infarction produced by occluding middle cerebral artery in stroke-prone spontaneously hypertensive rats. Gen Pharmacol 22:143–150

24.

O'Gorman

Sidman

(1985a) Degeneration of thalamic neurons in “Purkinje Cell Degeneration” mutant mice. I. Distribution of neuron loss. J Comp Neurol 234:277–297

25.

O'Gorman

(1985b) Degeneration of thalamic neurons in “Purkinje Cell Degeneration” mutant mice. 2. Cytology of neuron loss. J Comp Neurol 234:298–316

26.

Oldendorf

(1971) Brain uptake of radiolabeled amino acids, amines and hexoses after arterial injection. Am J Physiol 221:1629–1639

27.

Shigeno

Yamasaki

Kato

Kusaka

Mima

Takakura

Graham

Furukawa

(1990) Reduction of delayed neuronal death by inhibition of protein synthesis. Neurosci Lett 120:117–119

28.

Stumpf

Shughrue

Duncan

(1991) The use of autoradiography for the regional and cellular-subcellular localization of radiolabeled substances in the central nervous system. In, Neuroscience Research Methods, Vol. 2, ( Greenstein

, ed) Chur, Switzerland, Hardwood Academic Publishers, pp 631–652

29.

Tamura

Graham

McCulloch

Teasdale

(1981a) Focal cerebral ischaemia In the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1:53–60

30.

Tamura

Graham

McCulloch

Teasdale

(1981b) Focal cerebral Ischaemia in the rat: 2. Regional cerebral blood flow determined by (¹⁴C) iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1:61–69

31.

Tamura

Tahira

Nagashima

Kirino

Gotoh

Hojo

Sano

(1991) Thalamic atrophy following cerebral infarction in the territory of the middle cerebral artery. Stroke 22:615–618

32.

Tokuno

Kataoka

Asai

Chichibu

Kuroda

Ioku

Yamada

Hayakawa

(1992) Functional changes in thalamic relay neurons after focal cerebral infarct: a study of unit recordings from BPL neurons after MCA occlusion in rats. J Cereb Blood Flow Metab 12:954–961

33.

Yamada

Kinoshita

Kohmura

Sakaguchi

Taguchi

Kataoka

Hayakawa

(1991) Basic fibroblast growth factor prevents thalamic degeneration after cortical infarction. J Cereb Blood Flow Metab 11:472–478

34.

Yoshimine

Hayakawa

Kato

Yamada

Matsumoto

Ushio

Mogami

(1987) Autoradiographic study of regional protein synthesis in focal cerebral ischemia with TCA wash and image subtraction techniques. J Cereb Blood Flow Metab 7:387–393

35.

Watanabe

Kumon

Ohta

Nakano

Sakaki

Matsuda

Sakanaka

(1997) Protein synthesis inhibitor transiently reduces neuronal death in the thalamus following cortical infarction. Neurosci Lett 233:25–28

Changes in Protein Synthesis and Calcium Homeostasis in the Thalamus of Spontaneously Hypertensive Rats with Focal Cerebral Ischemia

Abstract

Keywords

MATERIALS AND METHODS

Animal model

Light microscopy

Electron microscopy

Measurement of rCBF

[14C]-leucine autoradiography

45Ca autoradiography

Statistical analysis

RESULTS

Light microscopy

Electron microscopy

[14C] iodoantipyrine autoradiography

L-[14C]-leucine autoradiography

45Ca autoradiography

DISCUSSION

Footnotes

Acknowledgments

Abbreviations used

References