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Lipids of the oligodendroglial fraction in methylnitrosourea induced encephalopathy
Exp. Path. 20, 64-67 (1981) Department of Neurology (Head: Prof. Dr. xl. WENDER) Academy of Medicine, Poznan, Poland
Lipids of the oligodendroglial fraction in methylnitrosourea induced encephalopathy By M. WENDER, Z. ADAMCZEWSKA-GONCERZEWICZ, A. GONCERZEWICZ, J. PANKRAC and D. TALKowsKA (Received October 20, 1980)
Address for correspondence,' Prof. Dr. M. WENDER, Department of Neurology, Academy of Medicine 49 Przybyszewskiego Str., 60-355 Poznan, Poland Key w or d s: lipids; oligodendroglia; methylnitrosourea (}1NU); encephalopathy; central nervous system; phospholipids; sphingomyelins; phosphatidylserine; myelin sheaths; rat
Summary The investigations were performed on oligodendroglial cells isolated from the cerebral white matter of Wistar rats treated intravenously with a 3 % solution of MNU at a dose of 60 mg! kg body weight. The oligodendroglial fraction showed a decrease of the sphingomyelin, phosphatidylserine and phosphoinositides content. The observed changes correlated with alterations of the lipid spectrum found in the myelin fraction of MNU intoxicated rats. This would indicate that MNU acting in the central nervous system affects in a similar way the lipid metabolism of both components of the oligodendroglia-myelin system.
Introduction Our previous studies (WENDER et al. 1977) have revealed that intoxication by methylnitrosourea (MNU) is capable of disturbing the lipid metabolism of membrane structures of the central nervous system such as the myelin sheaths. The most striking feature in this experimental condition, and so far not reported in typical demyelinating processes (WENDER and ADAMCZEWSKA 1974; NORTON 1977), was the massive loss of sphingomyelins from the affected sheaths. The other essential structural compound of the myelin sheaths - the proteins did not demonstrate any detectable changes of the protein profile in MNU-induced encephalopathy. The morphological study revealed spongious changes in the white matter, a picture that closely resembled that observed following triethyltin intoxication. The myelin sheaths form together with oligodendrocytes a biological compartment separate from the rest of the central nervous system. RUMS BY (1980) emphasized two points of interrelationships between these two components. A structural one, connected with antigenic properties, located at the external surfaces of the system, and a metabolic and dynamic one - with a principal localization in the oligodendroglial cell body. Hence a study concerning the influence of MNU intoxication on the lipid pattern of the oligodendroglial fraction in the encephalopathy evoked by this noxious agent seemed necessary and useful.
N1 aterials and 1\11ethods The investigations were performed on oligodendroglial cells isolated from the cerebral white matter of Wi star rats treated intravenously with a 3 % solution of ~[NU in 0.1 M MclLwAI~ citrate-phosphate
64
c·
~
~
23.5 ± 1.01 18.0 ± O.4B 49.0 ± B.BO
1.77 2.10 0.53 0.40 1.114 0.57 0.26 1.91
0.41
7.7 ± 0.5 49.5 ± 1.9 B.O ± 1.0 7.1 ± 0.9 16.S ± 1.1 11.4 ± 0.1 4.8 ± 0.9
13.6 ± 1.0 44.5 ± 2.8 B.1 ± O.S 7.6 ± 0.4
14.5 ± 0.5 11.1 ± 0.3 S.G ± 1.1 G
2 weeks
Normnl
Time nfter MNU intoxication
The vnlues are given as mean ± S.E. Number of experiments in each group Significnnt differences bold-faced
Sphingomyelins Phosphatidylehol ine Lysophosphatidylchol ine Phosphatidylserine + Phosphoinositides Phosphntidylethanolamine Plnsmnlogen N onidentified
Lipids
± ± ± ±
0.8 1.9 0.2 0.1 10.6 ± 1.1 10.S ± 1.2 G.1 ± l.B
13.B fi4.0 B.S 2.0
3 weeks
1.4 1.7 O.fi 1.4 12.9 ± 0.2 l1.S ± 0.6 7.1 ± 2.0
10.2 ± 50.5 ± 3.5 ± 4.3 ±
4 weeks
2.30 2.89 0.88 1.09
0.8B
21.8 ± 0.92 16.0 ± 0.84 S2.0 ± 2.74
24.9 ± traces 10.3 ± 79.9 ± 3.8 ± 6.4 ±
6 weeks
14.2 ± 0.9 10.7 ± 0.7 G.O ± 0.8
14.6 ± 0.2 10.7 ± 0.6 G.O ± 1.0
1.6
6.4 ± 0.8 5S.2 ± 1.5 B.O ± 0.2 4.5 ± 1.0
± ± ± ±
8 weeks 1.4 O.S 0.9
7.0 S3.6 2.S ii.6
6 weeks
1.24 B.52 0.B5 1.07
0.90
19.4 ± 0.82 14.8 ± 1.80 52.0 ± 2.2S
22.4 ± traces 8.8 ± 7fi.6 ± 4.1 ± 6.2 ±
8 weeks
% of total phospholipids)
16.1 ± 0.28 14.B ± 0.[)7 [)4.0 :l: 1.88
17.3 ± tm('es 12.7 ± 7,),1 :-!: 4.4 J: ii.1) ±
4 weeks
Table 2. Phospholipid composition of the cerebral oligodendroglinl fraction in MNU intoxication (in
22.4 ± 2.4fi 15.3 ± O.Gi) 5B.0 ± B.34
0.38 1.62
un
0.91
18.0 ± 2.20 17,fl ± 2.49 fiiLO ± 2.71
27.9 :l: 0.[)2 traecs 22.4 ± 0.90 80.B ± 1.16 5.9 :t: O.2() 3.4 ± 0.91)
0.8,)
17.3 ± traces 10.3 ± 77.2 ± fi.4 :±: 9.7 ±
24.1 ± traces 21.9 ± 72.1 ± fi.1 ± 12.fi ± 0.83
3 weeks
2 weeks
Normnl
Time after MNU intoxicntion
The vnlues are given ns menn ± S.E. Number of experiments in en£h group - () Signifimnt differences bold-faced
Cholesterol Cholesterol esters Sphingomyelins Phosphatidyleholine Lysophosphntidylchol ine Phosphntidylserine + Phosphoinositides Phosph
Lipids
Table 1. Lipid composition of cerebral oligodendroglial fraction in MNU intoxiention in mg/g of protein
± 1.12 ± 1.03 ± 2.23
0.44 2.29 0.35 1.05
0.27
± 0.9 ± 2.1 ± 0.1 ± 0.2 14.6 ± 0.3 9.1 ± 0.1 7.8 ± 1.6
8.1 51.2 3.4 5.8
12 weeks
15.4 19.5 52.0
24.0 ± tmces 9.0 ± 73.5 ± 3.5 ± 6.1 ±
12 weeks
buffer pH 5.8. The p oison was administered at dose of 60 mht. The olio-odendroo-lial fraction WitS isolated accordin g to the method of IQBAL and TELEZ:NAG:L (1972) as'" mo difiecl by FREYSZ et al. (1972). Totallipi(ls of the oligodendroglia] fra ction were extracted by means of the procedure described by F?L ClI~P [ et al. (1957) an? t~en furth er separated by means of thin layer chromatography as descnbed III onr prevlOus pubheatIOn (WE NDER and AD,DI CZEWSKA 19(4). Cholesterol was determined lI.sing the tl~ethod of SPERRY a nd WEBB (1950) a ~d the phospho r us content of the individual phospholipId fractlO ns by meanR of the method descnbed by BARTLETT (1959). The protein content was determined using the metho d of LOWRY et al. (1951).
Results
Results concerning the lipid composition of the oligodendroglial fraction of rats intoxicated by MNU in relation to its protein content are shown in table 1. Table 2 presents the percent composition of phospholipids. The results obviate significant differences of the oligodendroglial composition between the intoxicated rats and the normal animals. The most striking alterations were the decrease of both the absolute and relative content of sphingomyelins and fraction containing serinopllOsphatides and phosphoinositides in almost all experimental groups.
Discussion
The oligodendroglial fraction of almost all experimental groups showed a statistically significant decrease of sphingomyelin, phosphatidylserine and phosphoinositides content irrespect:vely of whether calculated per g of protein or whether expressed in percentages of total phospholipids. It was interesting to note that the observed changes in the oligodendroglial fraction correlated well with alterations of the lipid spectrum found in the myelin fraction of lIND intoxicated rats (WENDER et al. 1977). This very finding could indicate that MNU acting in the central nervous system affects in a like manner the lipid metabolism of both components of the oligodendroglia-myelin system, and it may in turn constitute the subcellular basis for the dem yelinating process occurring in these experimental condition (KROH 1973; KROH and CERvos-NAVARRO 1980). According to KROH (1980) who observed the appeara.n ce of nests of villous interfascicular oligodendroglia in animals treated with alkylnitrosourea compound, the unusual character of oligodendroglial changes might indicate some kind of neoplastic or reactive transformation resultipg in a loss of normal processes of oligodendroglial cells. The author suggested that ensueing demyelination is an event which is secondary to the primary transformation of oligodendroglia.
Litera/ure BARTLETT, G., Phosphorus assay in column chromatography. J. bioI. Chem. 234, 466-468 (1959). FOLCH-PI, J., J. LEES and G. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues. J. bioI. Chem. 226, 497- 511 (1957). FREYSZ, L., A. FAROOQUl, Z. ADAMCZEWS KA-GONCERZEWICZ and P. MANDEL, Lysosomal hydTo]ases in neuronal, astroglial and oligodendroglial enriched fra ct ions of rabbit and beef brain. J. Lipid Res. 20, 503-508 (1979). IQBAL, K., and J. TELLEZ-NAGE L, Isolation of neurons and gli a cells from normal and pathological human brain. Brain Res. 45, 296-301 (1972). KROll , H. , Experimental model of demyelination. 2nd Common Symposium of the Society of Neuropathology of the GDR and Association of Polish Neuropathologists. Erfurt 1980, p. 16. - Ethylnitrosourea induced microcephaly in Swiss mice and Wi star rats. In: Aktuelle Probleme der Neuropathologie (Ed. K. JELLINGER). Facultas Verlag, Wien 1973, pp. 29-35.
66
LOWRY, 0., N. ROSENBROUGH, A. FARR and R. RA:'iDALL, Protein measurement with the Folin phenol reagent. J. bioI. Chern. 193, 265-275 (1951). NORTON, W., Chemical pathology of diseases involving myelin. In: :iVIyelin (Ed. P. "MORELL). Plenum Press, New York and London 1977, pp. 383-414. RUMSBY, M., Oligodendrocyte-myelin sheat interrelationship. In: Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous System (Ed. A. BOESE). Verlag Chemie, Weinheim 1980, pp. 50-63. SPERRY, W., and M. WEBB, A revision of the Schoenheimer-Sperry method for cholesterol determination. J. bio!. Chern. 181, 97-106 (1950). WENDER, M., Z. ADAMCZEWSKA-GONCERZEWICZ, O. }IULAREK, J. PANKRAC and .J. SEDZIK, The effect of intoxication with methylnitrosourea on the lipid composition of cerebral myelin. Neuropat. Pol.
Vi, 219-230 (1977).
- , ,tnd Z. ADAMCZEWSKA, Lipidveranderungen im Gehirn be; Krankheiten, die mit Myelinschiidigung verbunden sind. In: Neue Forschungsergebnisse des Hirnstoffwechsels und der Entmarkungsenzephalomyelitis (Hrsg. R. SCHMIDT). Halle-Wittenberg 1974, pp. 248-255.
67
Lipids of the oligodendroglial fraction in methylnitrosourea induced encephalopathy By M. WENDER, Z. ADAMCZEWSKA-GONCERZEWICZ, A. GONCERZEWICZ, J. PANKRAC and D. TALKowsKA (Received October 20, 1980)
Address for correspondence,' Prof. Dr. M. WENDER, Department of Neurology, Academy of Medicine 49 Przybyszewskiego Str., 60-355 Poznan, Poland Key w or d s: lipids; oligodendroglia; methylnitrosourea (}1NU); encephalopathy; central nervous system; phospholipids; sphingomyelins; phosphatidylserine; myelin sheaths; rat
Summary The investigations were performed on oligodendroglial cells isolated from the cerebral white matter of Wistar rats treated intravenously with a 3 % solution of MNU at a dose of 60 mg! kg body weight. The oligodendroglial fraction showed a decrease of the sphingomyelin, phosphatidylserine and phosphoinositides content. The observed changes correlated with alterations of the lipid spectrum found in the myelin fraction of MNU intoxicated rats. This would indicate that MNU acting in the central nervous system affects in a similar way the lipid metabolism of both components of the oligodendroglia-myelin system.
Introduction Our previous studies (WENDER et al. 1977) have revealed that intoxication by methylnitrosourea (MNU) is capable of disturbing the lipid metabolism of membrane structures of the central nervous system such as the myelin sheaths. The most striking feature in this experimental condition, and so far not reported in typical demyelinating processes (WENDER and ADAMCZEWSKA 1974; NORTON 1977), was the massive loss of sphingomyelins from the affected sheaths. The other essential structural compound of the myelin sheaths - the proteins did not demonstrate any detectable changes of the protein profile in MNU-induced encephalopathy. The morphological study revealed spongious changes in the white matter, a picture that closely resembled that observed following triethyltin intoxication. The myelin sheaths form together with oligodendrocytes a biological compartment separate from the rest of the central nervous system. RUMS BY (1980) emphasized two points of interrelationships between these two components. A structural one, connected with antigenic properties, located at the external surfaces of the system, and a metabolic and dynamic one - with a principal localization in the oligodendroglial cell body. Hence a study concerning the influence of MNU intoxication on the lipid pattern of the oligodendroglial fraction in the encephalopathy evoked by this noxious agent seemed necessary and useful.
N1 aterials and 1\11ethods The investigations were performed on oligodendroglial cells isolated from the cerebral white matter of Wi star rats treated intravenously with a 3 % solution of ~[NU in 0.1 M MclLwAI~ citrate-phosphate
64
c·
~
~
23.5 ± 1.01 18.0 ± O.4B 49.0 ± B.BO
1.77 2.10 0.53 0.40 1.114 0.57 0.26 1.91
0.41
7.7 ± 0.5 49.5 ± 1.9 B.O ± 1.0 7.1 ± 0.9 16.S ± 1.1 11.4 ± 0.1 4.8 ± 0.9
13.6 ± 1.0 44.5 ± 2.8 B.1 ± O.S 7.6 ± 0.4
14.5 ± 0.5 11.1 ± 0.3 S.G ± 1.1 G
2 weeks
Normnl
Time nfter MNU intoxication
The vnlues are given as mean ± S.E. Number of experiments in each group Significnnt differences bold-faced
Sphingomyelins Phosphatidylehol ine Lysophosphatidylchol ine Phosphatidylserine + Phosphoinositides Phosphntidylethanolamine Plnsmnlogen N onidentified
Lipids
± ± ± ±
0.8 1.9 0.2 0.1 10.6 ± 1.1 10.S ± 1.2 G.1 ± l.B
13.B fi4.0 B.S 2.0
3 weeks
1.4 1.7 O.fi 1.4 12.9 ± 0.2 l1.S ± 0.6 7.1 ± 2.0
10.2 ± 50.5 ± 3.5 ± 4.3 ±
4 weeks
2.30 2.89 0.88 1.09
0.8B
21.8 ± 0.92 16.0 ± 0.84 S2.0 ± 2.74
24.9 ± traces 10.3 ± 79.9 ± 3.8 ± 6.4 ±
6 weeks
14.2 ± 0.9 10.7 ± 0.7 G.O ± 0.8
14.6 ± 0.2 10.7 ± 0.6 G.O ± 1.0
1.6
6.4 ± 0.8 5S.2 ± 1.5 B.O ± 0.2 4.5 ± 1.0
± ± ± ±
8 weeks 1.4 O.S 0.9
7.0 S3.6 2.S ii.6
6 weeks
1.24 B.52 0.B5 1.07
0.90
19.4 ± 0.82 14.8 ± 1.80 52.0 ± 2.2S
22.4 ± traces 8.8 ± 7fi.6 ± 4.1 ± 6.2 ±
8 weeks
% of total phospholipids)
16.1 ± 0.28 14.B ± 0.[)7 [)4.0 :l: 1.88
17.3 ± tm('es 12.7 ± 7,),1 :-!: 4.4 J: ii.1) ±
4 weeks
Table 2. Phospholipid composition of the cerebral oligodendroglinl fraction in MNU intoxication (in
22.4 ± 2.4fi 15.3 ± O.Gi) 5B.0 ± B.34
0.38 1.62
un
0.91
18.0 ± 2.20 17,fl ± 2.49 fiiLO ± 2.71
27.9 :l: 0.[)2 traecs 22.4 ± 0.90 80.B ± 1.16 5.9 :t: O.2() 3.4 ± 0.91)
0.8,)
17.3 ± traces 10.3 ± 77.2 ± fi.4 :±: 9.7 ±
24.1 ± traces 21.9 ± 72.1 ± fi.1 ± 12.fi ± 0.83
3 weeks
2 weeks
Normnl
Time after MNU intoxicntion
The vnlues are given ns menn ± S.E. Number of experiments in en£h group - () Signifimnt differences bold-faced
Cholesterol Cholesterol esters Sphingomyelins Phosphatidyleholine Lysophosphntidylchol ine Phosphntidylserine + Phosphoinositides Phosph
Lipids
Table 1. Lipid composition of cerebral oligodendroglial fraction in MNU intoxiention in mg/g of protein
± 1.12 ± 1.03 ± 2.23
0.44 2.29 0.35 1.05
0.27
± 0.9 ± 2.1 ± 0.1 ± 0.2 14.6 ± 0.3 9.1 ± 0.1 7.8 ± 1.6
8.1 51.2 3.4 5.8
12 weeks
15.4 19.5 52.0
24.0 ± tmces 9.0 ± 73.5 ± 3.5 ± 6.1 ±
12 weeks
buffer pH 5.8. The p oison was administered at dose of 60 m
Results
Results concerning the lipid composition of the oligodendroglial fraction of rats intoxicated by MNU in relation to its protein content are shown in table 1. Table 2 presents the percent composition of phospholipids. The results obviate significant differences of the oligodendroglial composition between the intoxicated rats and the normal animals. The most striking alterations were the decrease of both the absolute and relative content of sphingomyelins and fraction containing serinopllOsphatides and phosphoinositides in almost all experimental groups.
Discussion
The oligodendroglial fraction of almost all experimental groups showed a statistically significant decrease of sphingomyelin, phosphatidylserine and phosphoinositides content irrespect:vely of whether calculated per g of protein or whether expressed in percentages of total phospholipids. It was interesting to note that the observed changes in the oligodendroglial fraction correlated well with alterations of the lipid spectrum found in the myelin fraction of lIND intoxicated rats (WENDER et al. 1977). This very finding could indicate that MNU acting in the central nervous system affects in a like manner the lipid metabolism of both components of the oligodendroglia-myelin system, and it may in turn constitute the subcellular basis for the dem yelinating process occurring in these experimental condition (KROH 1973; KROH and CERvos-NAVARRO 1980). According to KROH (1980) who observed the appeara.n ce of nests of villous interfascicular oligodendroglia in animals treated with alkylnitrosourea compound, the unusual character of oligodendroglial changes might indicate some kind of neoplastic or reactive transformation resultipg in a loss of normal processes of oligodendroglial cells. The author suggested that ensueing demyelination is an event which is secondary to the primary transformation of oligodendroglia.
Litera/ure BARTLETT, G., Phosphorus assay in column chromatography. J. bioI. Chem. 234, 466-468 (1959). FOLCH-PI, J., J. LEES and G. SLOANE-STANLEY, A simple method for the isolation and purification of total lipids from animal tissues. J. bioI. Chem. 226, 497- 511 (1957). FREYSZ, L., A. FAROOQUl, Z. ADAMCZEWS KA-GONCERZEWICZ and P. MANDEL, Lysosomal hydTo]ases in neuronal, astroglial and oligodendroglial enriched fra ct ions of rabbit and beef brain. J. Lipid Res. 20, 503-508 (1979). IQBAL, K., and J. TELLEZ-NAGE L, Isolation of neurons and gli a cells from normal and pathological human brain. Brain Res. 45, 296-301 (1972). KROll , H. , Experimental model of demyelination. 2nd Common Symposium of the Society of Neuropathology of the GDR and Association of Polish Neuropathologists. Erfurt 1980, p. 16. - Ethylnitrosourea induced microcephaly in Swiss mice and Wi star rats. In: Aktuelle Probleme der Neuropathologie (Ed. K. JELLINGER). Facultas Verlag, Wien 1973, pp. 29-35.
66
LOWRY, 0., N. ROSENBROUGH, A. FARR and R. RA:'iDALL, Protein measurement with the Folin phenol reagent. J. bioI. Chern. 193, 265-275 (1951). NORTON, W., Chemical pathology of diseases involving myelin. In: :iVIyelin (Ed. P. "MORELL). Plenum Press, New York and London 1977, pp. 383-414. RUMSBY, M., Oligodendrocyte-myelin sheat interrelationship. In: Search for the Cause of Multiple Sclerosis and Other Chronic Diseases of the Central Nervous System (Ed. A. BOESE). Verlag Chemie, Weinheim 1980, pp. 50-63. SPERRY, W., and M. WEBB, A revision of the Schoenheimer-Sperry method for cholesterol determination. J. bio!. Chern. 181, 97-106 (1950). WENDER, M., Z. ADAMCZEWSKA-GONCERZEWICZ, O. }IULAREK, J. PANKRAC and .J. SEDZIK, The effect of intoxication with methylnitrosourea on the lipid composition of cerebral myelin. Neuropat. Pol.
Vi, 219-230 (1977).
- , ,tnd Z. ADAMCZEWSKA, Lipidveranderungen im Gehirn be; Krankheiten, die mit Myelinschiidigung verbunden sind. In: Neue Forschungsergebnisse des Hirnstoffwechsels und der Entmarkungsenzephalomyelitis (Hrsg. R. SCHMIDT). Halle-Wittenberg 1974, pp. 248-255.
67