Karyometric and cytophotometric studies of the oligodendroglia in the corpus callosum of the rat after hypoxia

Exp. Pathol. 1988; 33: 249-255 VEB Gustav Fischer Verlag J ena Department of Neurology (Head: Prof. Dr. med. M. WENDER), Medical School Poznan, Poland

Karyometric and cytophotometric studies of the oligodendroglia in the corpus callosum of the rat after hypoxia By M. WENDER, J. SZCZECH, A. GODLEWSKI and A. GROCHOWALSKA Address for correspondence: Prof. Dr. med. M. WENDER, Department of Neurology, School of Medicine, 49, Przybyzcwskiego Str. , 60-355 Poznan, Poland Key words: oligodendroglia; karyometry; DNA cytophotometry; hypoxia

Summary Karyometric and cytophotometric investigations were conducted using an automatic analyser "Morphoquant" on oligodendroglial nuclei of corpus callosum following acute or moderate hypoxia in Wistar rats. Results of our studies demonstrated karyometric changes in nuclei of oligodendroglia cells after hypoxia, with deviations distinct in the early and late period after exposure to hypoxia. Cytophotometric analysis of a relative DNA content in cell nuclei of oligodendroglia demonstrated an increased extinction of DNA in the early period and a decreased extinction in the late period after acute or moderate hypoxia. The changes in oligodendroglia may be primarily responsible for the molecular structure lesion in myelin, observed as a result of hypoxia.

Introduction Hypoxia and more rarely, ischemia, may lead to lesions of myelin sheaths, sometimes in the form of markedly disseminated or diffuse demyelination (RAINE 1984). The phenomenon is known from case descriptions in human pathology (PLUM et al. 1962; WENDER et al. 1964 ; TRAUGOTT and RAINE 1984) as well as from experimental studies. Principal experimental models used in the studies include carbon monoxide or cyanide intoxication, leading through acute hypoxia to demyelination, expressed mainly in the brain commisural system (HIRANO et al. 1967; WEND ER et al. 1978 and 1986). However, it is still unclear. whether hypoxia directly destroys the myelin or rather oligodendroglia represents the target structure whieh indirectly results in demyelination. VAN HUTTEN and FRIEDE (1963) and IBRAHIM et al. (1963) have advocat ed the ,iew that a metabolic lesion of oligodendroglia is the primary event in eyanide demyelination. However, studies of WENDER et al. (1972) as well as recently tho~e of FONCIN et al. (1986) have failed to confirm this statement. Therefore in our eomplex studies concerning the effect of hypoxia on cerebral white matter we have performed karyo- and cytophotometric investigations of oligodendroglia in the corpus callosum of rats SUbjected to hypoxic hypoxia.

Material and 111eihods Experiments were performed on Wistar rats, each weighing 200-250 g. The animals were placed for 3 min in a specially constructed glass chamber, into whieh a gaseous mixture of 2 % oxygen, 97.9 % nitrogen and 0.1 % car bon dioxid e (acute hypoxia) was introduced or they were placed for 30 min in a gaseo us mixture of 7 % oxygen, 92.9 % nitrogen and 0.1 % carbon dioxide. Appropriate composition of gases in the chamber was secured by feeding the gases through flowmeter and rotometer produ ced by Junkalor Dessau. Immediately after the end of hypoxia the mixed arterio-venous blood was t aken from the tail and the gasometric estimations of pH, p02 and pCO z were performed

16

249

on the BMS-2 apparatus produced by the Radiometer Company. The animals were sacrificed after 4 h, 24 h, 14 days and 2 months after the experiment. Results of experimental studies were compared with those for the control group of normal animals. The brains were instantly removed and fixed in the Baker solution at room temperature. Paraffin sections, 7 11m in thickness were stained with cresyl violet and according to Weil method. Other sections were subjected to Feulgen reaction for DNA. The acid hydrolysis time of 3.5 h at 37°C for Feulgen method was chosen using the hydrolysis curve established earlier, according to KRYGIER-STOJALOWSKA (1982). Morpho- and cytophotometric studies were carried out in monochromatic light, at 560 nm wavelength using the automatic analyser of microscopic images "Morphoquant" (VEB Carl Zeiss JENA, G.D.R.). The following parameters of examined cells were measured using the MOSAIK programme (Voss et al. 1979). KONL - length of nuclear circumference determined by the number of raster points covering circumference of an examined structure, KOFL - cross section area of cell nucleus expressed by the total number of raster points on and inside the circumference of an examined object, FOF A - shape index of cell nucleus representing quotient of squared circumference to the crosssection area, DMVH - elongation index determined by the quotient of minimal to maximal diagonals. An extent of elongation is inversely proportional to value of this index, FL VH - shape regularity index of cell nucleus determined by the quotient of a cross-section area to an area of a smallest octagon circumscribed on the cross-section of an examined object; it illustrates the extent of folding of the examined object surface, EXTS - sum of extinctions of all points of an examined object, expressed in working units used in cytophotometry, KOMP - index determining compactness of nuclear chromatin, ZNTR - index determining rate of chromatin concentration around centre of the nucleus. Results of measurements of oligodendrocytes in experimental animals were compared to those in control rats using Kolmogorov-Smirnov nonparametric test (KST). Observed changes were considered statistically significant at the level of p :<::: 0.05.

Results

Histopathological studies No alterations were disclosed in the white matter. In contrast to this multiple degenerative changes in neurocytes, mainly of the chronic or ischemic type were observed. Gasometric estimations Results of gasometric studies, used a:i a criterion of hypoxia, are presented in table 1. Morphometric studies Results of karyo- and cytophotometric estimations of oligodendroglia nuclei are presented in table 2.

Table 1. Results of gasometric estimations in the mixed blood immediately after completing exposure to experimental hypoxia pH Control group Acute hypoxia (2 % of oxygen in respiratory mixture during 3 min) Moderate hypoxia (7 % of oxygen in respiratory mixture during 30 min)

p0 2 (in mm Hg)

7.33

± 0.04

7.14

± 0.05**

75.8 40.6

± 4.3 ± 3.4**

41.8 62.9

± 3.5 ± 4.0**

7.14

± 0.06**

44.6

± 7.5**

53.5

± 3.5**

Mean from 10 estimations + S.E. ** Difference highly significmnt at the level p ~ 0.01

250

Exp. Pathol. 33 (1988) 4

pC0 2 (in mm Hg)

C> ......

t-:l

II>-

.$

C¥:J

~

-;:;

~ ~

~

::r

~

'"d

'"!='l

~

t:<:I

..... C":> *

60 d

4h

24 h

2 % O2

7 % O2

7 % O2

95 %

14 d

2% O2

KST KST KST KST

<

= 100 % = 99 % = 95 %

7% O2

p:S:;; 0.001 p:S:;; 0.01 p:S:;; 0.05 not significant

60 d

14 d

24 h

2% O2

7% O2

4h

x ± SD min. max. x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST x ± SD min. max. KST

Control

2% O2

Value

Group 85 ± 66 111 96 ± 78 115 100% 104 ± 82 130 100% 76 ± 58 97 100% 78 ± 65 96 100% 90 ± 57 113 100% 91 ± 57 114 100% 85 ± 68 110 88% 82 ± 64 104 90%

KONL 244 ± 40 158 355 295 ± 30 214 389 100% 342 ± 39 244 454 100% 181 ± 32 120 268 100% 223 ± 26 166 314 100% 282 ± 44 107 404 100% 283 ± 47 107 394 100% 282 ± 34 201 367 100% 228 ± 35 144 343 99% 149 ± 129 191 157 ± 132 200 100 % 159 ± 136 216 100 % 161 ± 128 215 100 % 142 ± 128 168 100 % 147 ± 124 218 15% 148 ± 128 215 0% 150 ± 131 206 0% 146 ± 124 199 100 %

FOFA

* Abbreviations in text

8

7

9

8

5

7

8

7

8

KOFL

12

12

13

11

7

18

14

13

11

69 ± 10 44 96 75 ± 10 48 97 100% 75 ± 10 50 97 100 % 61 ± 13 32 96 100 % 77 ± 9 49 96 100 % 75 ± 11 46 100 100 % 74 ± 11 49 97 100 % 71±11 43 97 86% 71±11 43 96 100 %

DMVH

Table 2. Results of karyometric studies of oligodendroglia in corpus callosum of rats after hypoxia

87 ± 2 79 94 87 ± 2 79 93 100% 87 ± 2 78 92 100% 84 ± 4 71 92 10% 88 ± 2 82 94 100% 82 ± 2 79 94 100% 88 ± 2 77 94 88% 88 ± 2 79 94 97% 88 ± 3 72 9G 10%

FLVH

106 100% 80 ± 60 106 100% 64 ± 34 81 100% 73 ± 57 88 100% 78 ± 38 102 10 % 76 ± 35 105 100% 73 ± 48 95 100% 77± 55 9G 100%

54

112 81 ±

7

9

10

10

5

(j

8

8

78 ± 12

54

EXTS

75 60 ± 4 48 71 100 % 57 ± 5 42 73 100 % 65 ± 3 55 75 15 % 62 ± 3 50 72 100 % 64 ± 4 42 75 100 % 63 ± 5 42 79 100 % 65 ± 5 49 81 91 % 65 ± 3 56 77 79 %

54

65 ± 3

KO MP

77± 6 57 91 62 ± 8 37 88 100 % 60 ± 11 31 91 100% 85 ± 6 49 98 100 % 70 ± 7 49 92 100 % 71±1O 2G 9(j 100% 68 ± 12 19 93 100 % 67 ± 9 27 91 100% 78 ± 9 44 94 82%

ZNTR*

Table 3. Cell nuclei of oligodendroglia in corpus callosum categorized into classes according to KU1<'L index value (section area of cell nucleus) of rats subjected to acute hypoxia (2 % of oxygen in gaseous mixture) - 24 h after the exposure Individual nuclei

Classes

(%)

Absolute number 0- 99 100- 139 140- 179 180- 219 220- 259 260- 299 300- 339 340- 379 380- 419 420- 459 460-2047

Control

Experimental

Control

Experimental

0 0 9 80 113 65 28 5 0 0 0

0 0 0 0 7 45 84 111 45 8 0

0.0 0.0 3.0 26.6 37.6 21.6 9.3 1.6 0.0 0.0 0.0

0.0 0.0 0.0 0.0 2.3 15.0 28.0 37.0 15.0 2.6 0.0

Table 4. Cell nuclei of oligodendroglia in corpus callosum categorized into classes according to value of EXTS (sum of extinctions of all points of an examined object, expressed in working units used in cytophotometry) of rats subj ected to acute hypoxia (2 % of oxygen in gaseous mixture) -14 days after exposure Individual nuclei

Classes

(%)

Absolute nnmber 0- 29 30- 39

40- 49

5060708090100-

110-

59 69 79 89 99 109

119

120- 2047

Control

Experimental

Control

Experimental

0 0 0 4 59 150 38

0 0 0 0 26 109

0.0 0.0 0.0 1.3 19.8 50.0 12.6 3.6 10.3 2.3 0.0

0.0 0.0 0.0 0.0 8.6 36.3 38.0 16.3 6.0 0.0 0.0

11

31 7 0

114 49 2 0 0

Length of nuclear circumierence (KONL) demonstrated a number of changes in the experimental groups. In the early period (4 and 24 h) after acute hypoxia (2 % of oxygen in the gaseous mixture) elongation of the circumference and increase of cross-section area were noted, whereas in the later period (14th and 60th day after hypoxia) shortening of KONL and decrease of cross-section a,rea were observed. In the experimental group of moderate hypoxia (7 % of oxygen in the respiratory mixture) significant changes (elongation of nuclear circumference) were observed only in the early period (4 and 24 h after hypoxia). In all periods in the experimental group an increase of cross-section area of nuclei was established. Deviation in the index of nucleus shape (FOF A), in the form of its increase was found only in the group of acute hypoxia. In contrast to this, DMVH values (elongation index) demonstrated changes indicating spheric transformation of cell nuclei in both basic experimental groups (ar-ute and moderate hypoxia). The index of shape regularity (FLVH) remained unchanged in the majority of experimental groups. Results of cytophotometric estimations of relative DNA content in oligodendroglia nuclei demonstrated numerous changes. As a result of acute hypoxia an increase of the sum of estimations of all points of an examined nuclei occurred in the early period (4 and 24 h after experimental hypoxia), whereas reciprocal observations were made, i.e. a decrease of EXTS

252

Exp. Pathol. 33 (1988) 4

values was noted in the later period (14 days after acute hypoxia). In groups of moderate hypoxia the changes of EXTS index were similar, although less pronounced. In many experimental groups of animals subjected to acute or moderate hypoxia some loosening of chromatin structure (decrease of KOMP value) as well as a shift of chromatin granules to the periphery of cell nucleus (lowering of ZNTR index) were demonstrated. The above presented changes in mean values of karyo- and cytophotometric estimations, listed in details in table 2, resulted also in absolute and relative alterations in categorization of individual cells to distinct size classes. Some examples of such shifts are presented in tables 3 and 4.

Discussion Several morphological and histochemical changes in oligodendroglia have been described as a result of hypoxia. In carbon oxide encephalopathy, parallel to the appearance of demyelinating foci, a marked oedema of mitochondria, as well as formation of some pseudo myelinic figures were noticed (KORTHALS et al. 1973 b). Also HOPPE (1974) has emphasized great sensitivity of oligodendroglia to action of th e poison similar to that of neurocytes. In tissue culture of glia cells KORTHALS et al. (1973a) have observed spongious changes in oligodendroglia in the form of vesicles, sharply demarcated from the surrounding cytoplasm of normal electron density, resulting from CO intoxication. However, sensitivity of oligodendroglia to action of hypoxia has not been unequivocally established in all tissue culture studies. For instance KRASNICKA et al. (1973) have observed no changes in the cells after a short anoxia. In experimental studies on cyanide encephalopathy in the rat IBRAHIM et al. (1963) have noticed that oligodendroglia of corpus callosum shows increased enzyme activity and undergoes hyperplasia and hypertrophy in the prodromal stage, before the onset of demyelination. The authors have suggested that the hyperactivity of oligodendroglia is a primary event in cyanide induced demyelination, and not a response to myelin breakdown. This has not been confirmed in our own studies, in which we have observed rather decreased enzyme activities inside corpus callosum and hyperactivity in oligo- and astroglia at the edge of demyelinating foci (WENDER et al. 1972). MOSSAKOWSKI and GAJKOWSKA (1976), studying electron microscopic patterns of glia lesions induced with cyanide in tissue culture, have observed increased micropinocytotic activity, swelling of cytoplasm, distension of endoplasmic reticulum channels, lesion of mitochondria and accumulation of dense bodies in both types of glia cells. In oligodendroglia damage of mitochondria has predominated whereas in astrocytes exponents of disturbed permeability of cellular membranes were mainly observed. The differences between changes noted in astro- and oligodendroglia are in agreement with those described by KORTHALS et al. (1973 a) after carbon monoxide intoxication, studied in in vitro conditions. The severe lesion of oligodendroglial mitochondria contrasting with the very mild one in astroglia support the concept of YBATA et al. (1971) about the metabolic divergence of mitochondria in the two types of neuroglia. Similar results have been obtained by MOSSAKOWSKI (1978) who studied the glia lesion in tissue culture, provoked by sodium cyanate. The observed alterations of oligodendroglia consisted of cell swelling and vesicular degeneration, probably indicating disturbances in cell membrane permeability. From these studies the author has concluded that lesion of oligodendroglia plays a crucial role in the pathogenesis of cyanide demyelination. However, he has failed to explain selectivity of the changes, limited to the commisural systems. Recently, FONCIN et al. (1986) have returned to the problem underlying ultrastructural similarity of myelin changes in cyanide induced encephalopathy and in triethyltin sulphate intoxication, including an absence of changes in oligodendroglia in the early period. Lesions of these cells were observed only in the late period accompanying macrophage reaction. Exp. Pathol. 33 (1988) 4

253

In studies presented heretofore we have noted karyometric and cytophotometric deviations of oligodendroglia nuclei in acute and moderate hypoxia related to the morphological and histoenzymatic changes described in the literature in several experimental models of hypoxia. Enlargement of cell nuclei observed in the early period after hypoxia seems to reflect metabolic stimulation, whereas some nuclear shrinkage seen in the later period may be interpreted as sign of degeneration. Results of cytophotometric estimations showing concomitant changes in relative DNA estimation values are consisted with the conclusion. The latter may be interpreted only as reflecting changes in DNA configuration in oligodendroglia nuclei associated with some shift between the metabolically more active loose, and less active, heterochromatin DNA fractions. It is interesting to note that despite of marked deviations in oligodendroglia we have found no demyelination but only some changes in chemistry of myelin lipids. The latter should be interpreted as indicating that acute or moderate hypoxia only leads to a lesion of molecular myelin structure (WENDER et al. 1987) without evident myelin decay. However, it remains difficult to solve the basic question if the chemical changes in myelin reflect immediate effects of oxygen deficiency on metabolism of membraneous structure in myelin sheath, or rather represent a secondary effect of hypoxic lesions in oligodendroglia as indicated by our results. The described karyometric and cytophotometric changes in cell nuclei of oligodendroglia cannot be considered specific for the sequelae of hypoxia since similar, although not identical deviations have been observed after intoxication of rats with vincristine (KOZIK et al. 1982) and lomustine - CCNU (WYGLADALSKA-JERNAS et al. 1984).

References FONCIN, J., PERRE, N., RAJADFETRA, N.: Cyanide lencoencephalopathy: an experimental study with transmission electron microscopy. X. Intern. Congress Neuropathol., Stockholm 1986, Abstracts, 212. HIRANO, A., LEVINE, S., ZIMMERMAN, H.: Experimental cyanide encephalopathy. Electron microscopic observation of early lesions in white matter. J. Neuropathol. Exp. Neurol. 1967; 26: 200-213. HOPPE, B.: Obraz zmian strukturalnych i histochemicznych gleju hodowanego in vitro poddanego dzialniu tlenku wegla. Neuropat. Pol. 1974; 12: 371-384 (in Polish). HUTTEN, W. VAN, FRIEDE, R.: Histochemical studies of experimental demyelination produced with cyanide. Exp. Neurol. 1963; 4: 402-412. IBRAHIM, M., PHILANDER, E., BRISCOE jr., P., BAYLISS, 0., ADAMS, C.: The relationship between enzyme activity and neuroglia in the prodromal and demyelinating stages of cyanide encephalopathy in the rat. J. Neurol. Neurosurg. Psychiatry 1963; 26: 479-486. KORTHALS, J., HOPPE, B., KARWACKA, H.: Electron microscopic studies on the toxic effect of carbon monoxide on the glial tissue cultured in vitro. Neuropat. Pol. 1973a; 11: 315-322. - MOSSKOWSKI, M., SMIALEK, M.: Histological and electron microscopic picture of experimental carbon monoxide encephalopathy. N europat. Pol. 1973 b; 11: 279-300. KOZIK, M., SZCZECH, J., GODLEWSKI, A.: Karyometric studies and quantitation of the DNA content in nerve and oligodendroglial cells after intraperitoneal administration of vincristine. Acta histoc hem. 1982; 71: 175-182. KRASl'IICKA, Z., REKAWEK, K., GAJOWSKAJA, B.: Effect of short-term anoxia on the ultrastructural picture of the glial tissue in vitro. Neuropat. Pol. 1973; 11: 399-404. KRYGIER-STOJALOWSKA, A.: Principle of cytophotometrv. In: Topochemical methods in cell and tissue studies. PWN, Warszawa 1982, pp. 114-134. " MOSSAKOWSKI, M., GAJKOWSKA, B.: Electron microscopic picture of glia lesions induced with cyanide in tissue culture. Neuropat. Pol. 1976; 14: 441-449. - Morphology and histochemistry of glia alterations evoked by sodium cyanate in tissue culture. Neuropat. Pol. 1978; 16: 53-63. PLu)I, F., POSNER, J., HEIN, R.: Delayed neurological deterioration after anoxia. Arch. Intern. Med. 1962; 110: 18-25. RAINE, C.: The neuropathology of myelin diseases. In: MORELL, P. (ed.), Myelin. Plenum Press, . New York and London 1984, pp. 259-310. THAUGOTT, D., RAINE, C.: The neurology of myelin disease. In: MORELL, P. (ed.), Myelin. Plenum Press, New York and London 1984, pp. 311-335.

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Voss, K. , NEUMANN, E., WITSACK, W.: " Universelles Programmsystem" fiir den automatischen . Mikrobildanalysator Morphoquant. J enaer Rundsch. 1979; 24: 167-169. WENDER, M. , JURCZYK, W., STENGERT, K.: Cerebral lipids in myelinopathy caused by cardiac arrest. Acta Neuropath. 1964; 4: 238-244. . _ KOZIK, M., WAJGT, A.: Ristoenzymology of experimental cyanide encephalopathy. Acta his tochem. 1972; 43: 361-371. _ AD AMCZEwSKA-GONCERZEWICZ, Z. , WAJGT, A.: Myelin lipids in cyanide encephalopathy. Neuropat. Pol. 1978; 16: 153-162. _ _ STANISLAWSKA, J., KNITTER, B. , TALKowsKA, D., PANCRAC, J.: Influence of cyanide intoxication on the composition of myelin lipids in rats fed during dev elopment on a diet containing various amounts of lipids. Neuropat. Pol. 1986; 24: 43-56. _ _ _ PANKRAC, J., TALKOWSKil , D., GROCHOWALSKA, A.: Myelin lipids of the rat brain in experimental hypoxia. Exp. Pathol. 1988; 33: 59-63. WYGLADALSKA-JERNAS, R., SZCZECH, J., GODLEWSKI, A.: Cytophotometry of nucleic acids and morphometry of motor neurons and oligodendroglia in the spinal cord of rats treated with CCNU (Lomustine). J. Hirnforsch. 1984; 20: 487-492. YBATA, Y. , PICCOLI, F. , PAPPAS, G., LAJTHA, A.: An electron micros copic and biochemical study on the effect of cyanide and low Na + on rat brain slices. Brain Res. 1971; 30: 137-158. (Received March 18, 1987; Revised June 23, 1987)

Exp. Pathol. 1988; 33: 255-256 VEll Gustav Fischer Verlag J ena

Book Reviews Fetal and Neonatal Pathology edited by J. W. KEELING. 590 pages, with 587 figures, DM 298, - , ISBN: 3-540-16211-9. Springer-Verlag, Berlin- Heidelberg-N ew Y ork-London-Paris-Tokyo 1987. Recently neonatal paediatrics more and more has emerged as a field for specialization. This ineludes the performance of perinatal necropsies and fetal examination in order to provide satisfactory answers for clinicians regarding their day-to-day problems, but also directed to fundamental questions. Perinatal mortality continues to fall in Europe, North America and Australia but particular attention is directed to those deaths which still occur. - In this context the book is primarily intended for the histopathologist who is faced with perinatal necropsy service, and it will help to achieve competence in the performance of perinatal necropsies and fetal examination. Likewise the book is of high importance for obstetricians and neonatologists, because postmortem examination may be very useful for clinical diagnosis and therapy as well as for parental counselling about future pregnancies. The first part of the book (chapters 1-15) comprises important problems in fetal and neonatal pathology and is introduced by two chapters dealing with the perinatal necropsy and the normal implantation and placentation in the human involving the methods of placental examination, the findin gs under normal and abnormal conditions as well as specific pathological states. The following 4 chapters describe and discuss particular aspects of fetal pathology in the first and second trimesters of pregnancy, covering spontaneous abortions, prenatal diagnosis and pathological features of congenital abnormalities, and genetic metabolic diseases. - The remaining chapters of the first part arc largely devoted to problems of the second half of pregnancy, introdu ced by a ehapter about epidemiologk al analyses and continued by problem-orientated contributions covering the most essential clinical aspects, e.g. prematurity, intrapartum asphyxia and birth trauma, fetal hydrops, congenital tumors, infections, iatrogenic diseases, immunology under normal and pathological conditions, etc. The second part of the book (chapters 16-28) is system-orientated and covers the diseases of the gastrointestinal tract and exocrine pancreas , of the liver and gall bladder, the respiratory system, the cardiovascular system, the urogenital system, the endocrine system, the reticuloendothelial system and blood, of the central nervous system, the peripheral nerves and skeletal muscles, the skelet al system, the skin and of the eye and ear.- Each chapter is introduced by a concise description of the normal development of the particular system and the normal function in the neonatal prriod as well as -if it is relevant - by a presentation of the change. which take place at birth. Exp. Pat hoI. 33 (1988) 4

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