Cell Tiss. Res. 150, 369--375 (1974) �9 by Springer-Verlag 1974

Dissolution and Removal of Neuronal Lipofuscin Following Dimethylaminoethyl

p-Chlorophenoxyacetate Administration to Guinea Pigs

Mahdi Itasan*, Paul Glees, and Polly E. Spoerri* *

Institute of Histology and Neuroanatomy, University of GSttingen, Germany

Received iVfarch20, 1974

Summary. Dimethylaminoethyl p-chlorophenoxy acetate (80 mg/kg body weight) was administered (i. m.) to guinea pigs for 30 to 56 days. Electron microscopic examination of the hippocampus, mid-brain reticular formation and the area postrema revealed marked dimi- nution in the electron density of the pigment granules and vacuolization. This type of lipofusein was detected in some phagocytic cells and in the capillary endothelium. Conspicuous vacu- olization of the capillary wall was discernible. These changes were not observed in the "control group" of animals.

Key words : Lipofuscin - - Phagocytosis - - Vacuolization - - Capillary endothelium - - Electron microscopy.

Introduction

Chemical interference with the processes of aging is a comparat ively new field of enquiry. Al though significant a t tempts have been made in the recent years to decelerate physiological aging by chemical intervention, the mechanism of action of these drugs remains ill-understood. Lipofuscin pigment has been used as a "morphological indicator" for determining the efficacy of therapeutic agents. This choice is obvious since the increasing intraneuronal accumulat ion of lipo- fuscin is generally accepted as the most consistent cytological change correlated with the aging of mammals (see review, Hasan and Glees 1972a). N a n d y and Bourne (2966) observed tha t meclofenoxate (centrophenoxine), a drug used in the t rea tment of preseuile and senile confusion states, decreased brain lipofuscin in old guinea pigs. Marked elimination of the pigment was noticeable in animals t reated for 22 weeks. These findings were later confirmed by the histological studies of Chemnitius et al. (1970) on the nucleus gigantocellularis of the rat. Meier and Glees (1972) demonstrated, for the first time, the vacuolization of the pigment granules of the rat spinal ganglia following 4-8 weeks t reatment . But the ult imate fate of the pigment granules has, as yet, not been elucidated.

The aim of the present investigation is to s tudy the effect of dimethylamino- ethyl p-chlorophenoxyacetate on the lipofuscin in some selected regions of the brain (e.g. hippocampus, midbrain reticular formation and the area postrema) of the guinea pig. Furthermore, an a t t empt to follow the mode of removal of the

* Fellow of the Alexander yon Humboldt Foundation, on leave of absence from J. N. Medical College, A.M.U. Aligarh 202001, India. ** Recipients of "Deutsche Forschungsgemeinschaft" Grant No. 28/19.

370 M. Hasan et al.

p igment has been made. I n the h ippocampus , l ipofuscin accumula t ion begins a t an earl ier age and i t accumula tes more p igment per sect ion volume of the cerebral cor tex t h a n the other regions of the bra in (Reichel et al., 1968; Hasan and Glees 1973a). Fur the rmore , i ts invo lvement in h u m a n presenile and senile dement ias (Hirano and Z immerman , 1962) justifies thorough inves t igat ion. According to Glees and Griff i th (1952) the des t ruc t ion of h ippocampus leads to loss of m e m o r y and confusion. Also the re t icu lar fo rmat ion and area pos t rema are known to accumula te significant amoun t of p igment a t an ear ly age (Wilcox 1959; H a s a n and H e y d e r 1974).

Materials and Methods Ten guinea pigs over 3 years old were used for this study. They had been maintained two

in a cage at a temperature of 26~ ~: 3~ and humidity (50-55%). The pair put together in a cage were of the same age, sex and weight. They were weighed at the beginning and end of the experiment. While one of the pair was given intramuscular injections of dimethyl- aminoethyl p-chlorophenoxyacetate HC1 (available as "Helfergin") Promonta Labs. Ham- burg, 80 mg/kg body weight daily for 30 to 56 days, the other one received an equal volume of physiological saline per i. m. at the same time. Care was taken to keep the environmental conditions constant for the experimental and control group of animals. The "treated" and "control" guinea pigs were killed two at a time and the tissues were processed together. The animals were anaesthetized with intraperitoneal injection of sodium pentobarbital (Nembutal, Abbot) and perfused through the heart with phosphate-buffered paraformaldehyde/ghitaralde- hyde solution (pH 7.3) prepared as recommended by Karnovsky (1965). The brains were dissected and small pieces of hippocampus, area postrema and midbrain tegmentum were immersed in 6 % gluraraldehyde in phosphate buffer for 3 hours at 4 ~ C. Following a brief rinse in phosphate buffer (pH 7.3), the post-fixation was done in 1:1 mixture of 2% osmium tetroxide and 0.2 M buffer saccharose for 2 hours at 4~ Dehydration was carried out in graded alcohol and propylene oxide and the tissues were embedded in Epon 812. Ultrathin sections were cut with an LKB ultratome and "on grid" staining was done with lead citrate after Reynolds (1963). The sections were examined and photographed with a Zeiss 9S-2 electron microscope.

Results

Although the animals looked otherwise hea l thy , a loss of weight was no ted a t the end of the per iod of drug admin i s t r a t ion in all the cases (in one ins tance even up to 450 gms).

The general morpho logy of l ipofuscin granules in the "con t ro l " groups of animals conformed to the descr ip t ion a l r eady given by Hasan and Glees (1972b), H a s a n and Glees (1973a, b) and H a s a n and H e y d e r (1974). Most of the poly- morphic p igment granules were mingled wi th the mi tochondr ia l profiles in the vi- c in i ty of the nucleus (Fig. 1). Af ter 4 weeks of drug adminis t ra t ion , the p igment masses appeared to lose thei r e lectron dens i ty and large electron-lucid areas (vacuoles) became appa ren t (Fig. 2). Pa r t i cu la r ly no t ewor thy was the incorpora t ion of a few a l te red l ipofusein granules (Fig. 3) in the pe r ika ryon of react ive "phago- cyt ic" cells, rich in cy toplasmic organelles and exhibi t ing increased electron dens i ty (Fig. 4). Occasionally, these cells were observed close to capil lar ies (Fig. 5). I t m a y be no ted t h a t the fine s t ruc ture of capil lar ies in the t issues ob ta ined f rom the control animals conformed to the s t a n d a r d descr ip t ion (see Dahl 1973). The endothe l ium formed a complete cont inuous layer wi th no fenest ra t ions and d id not conta in vacuola ted lipofuscin. I n this context , the occurrence of a l te red

Dissolution and Removal of Neuronal Lipofuscin 371

~'ig. 1. Electron micrograph showing par t of a reticular neuron. Note the dense lipofuscin granules (arrow, L). x 25500

Fig. 2. Pa r t of a hippocampal neuron. Fading of electron density of lipofuscin granule (arrow, L) after 4 weeks of t rea tment , x 51000

Fig. 3. Drug-induced vacuolization in lipofuscin granules (arrow L) observed in a "dense phagocytic cell" of the hippocampus. X51000

25 Cell Tiss. ~Res. 150

Fig. 4. Altered lipofuscin (arrow, L) within the perikaryon of a "phagocyte" in the hippo- campus of a t reated guinea pig. N, cell nucleus. X 30000

Dissolution and Removal of Neuronal Lipofusein 373

lipofuscin in pericytes of treated animals (Fig. 6) and in the endothelial cell (Fig. 7) was remarkable. Vacuolated pigment granules were often lodged in large spaces within in the capillary endothelium. Furthermore, the occurrence of a series of vacuoles in the capillary endothelium of guinea pigs treated for 35 days and more merits particular attention (Fig. 8). I t appears tha t the number of endothelial vacuoles increases following longer duration of the drug administration (Fig. 9).

Discussion The loss of weight at the end of a period of dimethylaminoethyl p-chloro-

phenoxyacetate administration is also noted in a recent report by Hochschild (1973). This investigator has recorded loss of weight in albino mice following a drug administration, but as the drug increased the median, mean and maximum survival time from the start of drug administration by 29.5 %, 27.3 % (p : 0.039) and 39.7 % respectively, the conclusion was reached that the loss of weight is by no means a side-effect of this drug.

No information is so far available on the ultrastructural changes in different areas of the brain following injection of dimethylaminoethyl p-chlorophenoxy- acetate. The observations by Meier and Glees (1971) concerning the removal of lipofuscin by satellite cells of spinal ganglion cells cannot be compared to the conditions prevailing in the brain itself as central neurons possess no such active satellite cells (Glees and Gopinath, 1973). This difference is of particular relevance in view of the reported regional variation in the time sequence of appearance of the pigment (Wilcox 1959; Hasan and Glees, 1972a) and in the pigment architecto- nics (Braak, 1971). Earlier light microscopic study of Nandy and Bourne (1966) led to the assumption that as a result of the t reatment the pigment granules occupied smaller and smaller areas in the cytoplasm as if gradually shrinking away. This can only be explained, in terms of electron microscopy, on the basis of the diminution in electron density and increased vacuolization of the pigment granules.

The findings presented in this report are strongly suggestive of the partici- pation of the capillary endothelium in the removal of altered lipofuscin. The mode of transport of the pigment granules to the capillary endothelinm is, however, debatable. Whereas Spoerri and Glees (1973) have observed the removal of the pigment to the extracelhilar space by means of cytoplasmic protrusions, our present observations reveal the participation of phagocytic cells as well. Tissue culture studies in our laboratory by Spoerri and Glees (1974, in press) support this view, for these authors see an active intake of lipofuscin granules in proliferating endothelium cells of residual capillaries. In this connexion the origin of phagocytic cells or microglia from endothelial cells of capillaries, extensively discussed by Glees (1955, 1972) has to be stressed.

Acknowledgement. Our thanks are due to Mssrs. Promonta Labs (Hamburg) for the gift of Helfergin (Dimethylaminoethyl p- ehlorophenoxyacetate). We wish to thank Miss E. Heyder, Mr. J. Kirchhoff and Mr. W. Dresp for their excellent technical assistance. We are grateful to Mr. 1%. Dungan for the photographic work and to Mrs. M. del Carmen Weinrichter for the meticulous typing of the manuscript.

Fig. 5. Phagocytic cell (N, nucleus). Altered lipofuscin (arrow, L) is found in close proximity to a capillary of the hippocampus; en, endothelial nucleus, x 18000

25*

Fig. 6. Altered lipofuscin (arrow, L) in the pericyte of a capillary. Area postrema of a treated guinea pig; pn, perieyte nucleus; e, endothelium. • 51000

Fig. 7. Altered ]ipofuscin (arrow, L) in the endothelium (e) of a capillary in the hippocampus. ( • 30000); r, red blood cell. • 30000

Fig. 8. Capillary endothelium in the hippocampus. Note the linear row of vacuoles (V). • 25 500

Fig. 9. Capillary endothelium studded with a large number of vacuoles (V). Par t of a hippo- campal capillary. •

Dissolution and Removal of Neuronal Lipofuscin 375

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Prof. Dr. med. Paul Glees Institute of Histology and Neuroanatomy University of GSttingen 3400 GSttingen Kreuzbergring 36 Federal Republic of Germany