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HISTOCHEMICAL OBSERVATIONS ON OXIDATIVE ENZYME ACTIVITY I N REACTIVE MICROGLIA AND SOMATIC MACRO- PHAGES

BARBARA SMITH* and L. J. RUBINSTEIN? Department of Pathology, Maida Vale Hospital, and the Bernhard Baron Institute

of Pathology, The London Hospital

(PLATES CLIV AND CLV)

In a recent histochemical study, by the tetrazolium salt technique, of experi- mental cerebral edema produced by the local application of cold, it was noted that the fat-granule cells in the superficial cortical lesion displayed an enzymic response that differed constantly from that observed in the reactive astrocytes or the protein-carrying microglia in the edematous white matter (Rubinstein et al., 1962). The possibility of this being linked in some way to local response to tissue necrosis deserved further investigation and has prompted us to compare the histochemical changes displayed in a necrotic lesion by the activated microglia with those of the macrophage system elsewhere in the body under similar condi- tions. Attempts have also been made to activate the reticulo-endothelial system in regional lymph-glands and in the spleen by the local and systemic injection of antigens in order to ascertain whether the changes are similar to those obtained in the presence of necrotic tissue.

MATERIAL AND METHODS

Thirty adult rats of Wistar strain, weighing 150-200 g., and five 3-day-old rats were used. Lesions were produced in skeletal muscle, spleen and kidney by the application of solid carbon dioxide (dry ice) to the exposed tissue for 30 sec. This resulted in a sharply circumscribed area of necrosis. The cerebral lesions were made by applying the dry ice to the surface of the intact skull for 90 sec. ; this produced a saucer-shaped lesion usually confined to the cortex, but occasionally just encroaching on the white matter. Cerebral lesions were also made on the five 3-day-old rats. Most animals were killed after 5 or 6 days. In addition, 3 rats were given 0.5 ml. T.A.B. vaccine intravenously in order to activate the reticulo-endothelial system of the spleen without tissue damage, and killed 3 days later, and 3 others were given the same dose subcutaneously into the thoracic wall in order to activate the axillary lymph-glands, and killed 5 days later.

The fresh tissues were placed on chucks, which were then frozen rapidly between two pieces of dry ice. Tissues were cut on to coverslips in a Slee-Pearse cryostat, the brains a t I6 p and the other tissues at 8 p. The incubating mixtures containing the substrate and the tetrazolium salt were added to the coverslips supported over a waterbath at 37' C.

* Holder of M.R.C. Fellowship in Clinical Research. Present address : Department

t Present address : Laboratory Division, Montefiore Hospital, New York, 67. of Pathology, St. Bartholomew's Hospital, London, E.C.1.

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FIG. 1.-Necrotic lesion in muscle. Droplets of sudan- positive fat within macro- phages appear black. Col- loidal sudan I11 and IV. x 100.

FIG. 2.--Necrotising cortica cerebral lesion in adult rat. Reactive microglia, with intense formazan depo- sition. TPN diaphorase. x 210.

FIG. 3.-Same lesion as in fig. 1. Formazan deposition along necrotic fibres. TPN diaphorase. x 100.

ENZYME ACTIVITP I N MICROGLIA AND MACROPHAGES 573

The constituents of the incubating solutions and the conditions of incubation were identical with those described by Rubinstein et al. (1961). The enzymes and coenzymes investigated included diphosphopyridine nucleotide (DPN) diaphorase (cytochrome c reductase), triphosphopyridine nucleotide (TPN) diaphorase, and the dehydrogenases of glucose-6-phosphate, succinate, glutamate, the non- nucleotide dependent alpha-glycerophosphate, DPN-dependent isocitrate and TPN-dependent isocitrate. Incubations for succinate and alpha-glycerophos- phate dehydrogenase activity were effected in the presence of added menadione (Wattenberg and Leong, 1960). In addition to nitro-B.T. [2,2’-dipnitro- phenyl - 5,5’- diphenyl - 3,3’- (3,3‘- dimethoxy - 4,4‘- biphenylene) ditetrazolium chloride], M.T.T. [3 - (4,5 - dimethyl - thiazolyl- 2) - 2,5 - diphenyl tetrazolium bromide] chelated with cobaltous chloride was used in a number of procedures as the tetrazolium salt, according to the technique of Pearse (1960) ; in the latter case, sections were mounted, directly after incubation and formalin fixation, in glycerine-jelly : those treated with nitro-B.T. were rapidly dehydrated, cleared in xylol and mounted in D.P.X.

Post-fixed, fresh frozen sections or sections from paraffin-embedded blocks were stained with colloidal sudan I11 and IV (Govan, 1944), sudan black B, gallocyanin (Beswick, 1958), methyl green-pyronin, or hzematoxylin and eosin, or by the periodic acid-schiff (P.A.S.) or Weil-Davenport silver carbonate method. Some blocks were cut serially, in order to compare the histochemical findings more closely with the sudan and silver carbonate stains.

RESULTS The necrotic lesions in the brain, including those in the 3-day-old rats, and

in the spleen, kidney and muscle, contained a large number of cells with alcohol- soluble sudanophilic material in their cytoplasm (fig. 1). There was much more visible fat in sections stained with colloidal sudan I11 and IV than with the same dyes dissolved in 70 per cent. alcohol. These cells demonstrated the typical morphology and metalophil properties of reactive macrophages (Marshall, 1956, p. 23). A few macrophages appeared to contain a little P.A.S.-positive material; gallocyanin and methyl green-pyronin stains showed them to be devoid of ribonucleic acid. In the brain and spleen the phagocytic cells lay in a semi-circular layer in the periphery of the lesion nearest to the normal tissue; in the muscle they lay in parallel rows inside the dead fibres, and in the kidney, which has a poor macrophage response to necrosis (Marshall, 1956, p. 38), they were scattered sparsely about the necrotic area.

When studied in histochemical preparations, these macrophages were strongly positive for TPN diaphorase (figs. 2 and 3), TPN-dependent isocitrate dehydrogenase (figs. 4 and 5), and the non-nucleotide-dependent alpha-glycero- phosphate dehydrogenase, less strongly positive for glucose-6-phosphate dehydro- genase, and negative or very weakly positive for DPN diaphorase, DPN-dependent isocitrate, glutamate, and succinate dehydrogenases. By contrast, the reactive astrocytes a t the edge of the cerebral lesions, as well as the overlying meninges and blood vessels, were most strongly positive for DPN diaphorase (fig. 6), glutamate dehydrogenase and alpha-glycerophosphate dehydrogenase, though also moderately positive for TPN diaphorase and the TPN-dependent dehydrogenases. I n the normal muscle away from the lesions, as described by Dubowitz and Pearse (1960), only a proportion of the fibres stained strongly with the tricarboxylic acid cycle enzymes, i.e. succinate and DPN-dependent isocitrate dehydrogenases, and DPN diaphorase.

The formazan deposits in both M.T.T. and nitro-B.T. preparations, whether mounted in glycerine-jelly or D.P.X., appeared as small droplets in the cytoplasm of the cell. Because these droplets varied in size and seemed very similar to the fat droplets in the sudan preparations, they were thought not to be mitochondria,

574 BARBARA S M I T H AND L. J . RUBINSTEIN

but probably formazan adsorbed to the lipid, in which M.T.T. formazan is very soluble, although nitro-B.T. formazan is said not to be (Pearse, 1960).

In the normal spleen there were scattered TPN-diaphorase-positive cells, but there was no significant alteration in their size and number in the activated spleen of animals in which T.A.B. vaccine had been injected intravenously 3 days previously. The very enlarged axillary lymph-glands showed, in one section only, a small group of cells markedly positive for TPN diaphorase. The reticulo-endothelial cells activated by the injection of antigen did not otherwise show the histochemical changes demonstrated by similar cells in necrotic tissue.

DISCUSSION Macrophages containing sudanophilic fat have been described at the margins

of infarcts of many organs (Virchow, 1860 ; Karsner and Dwyer, 1916). This has usually been regarded as evidence of phagocytosis of cellular fat resulting from fatty degeneration. In the brain the sudanophilic material is often thought to be derived from myelin breakdown. It is, however, easily demonstrated in necrotic lesions in the adult rat cortex, which contains very little stainable myelin, and also in lesions in the immature rat brain before myelination occurs.

The histochemical results presented here are of interest since they demon- strate, in macrophages reacting to tissue breakdown in brain, muscle, spleen and kidney, an increased activity of enzymes known from in-vitro studies to take part in fatty acid synthesis. The findings are that the macrophages located in areas of necrosis show intense TPN-diaphorase and TPN-linked dehydrogenase activity. This response contrasts with that found in a reactive process not intimately associated with necrosis, such as astrocytic gliosis, where there is a marked increase of DPN diaphorase and DPN-linked dehydrogenase activity, and also with the findings in reticulo-endothelial cells activated by antigen injection, where no marked enzymatic change could be demonstrated.

TPN-dependent enzymes take part in at least two major metabolic pathways -fatty acid synthesis, and the hexose monophosphate shunt.

Porter et al. (1957) and Lachance et al. (1958) among others have shown in in-vitro studies that fatty acid synthesis is effected through a TPN-dependent system, microsomal in situation, in contrast to fatty acid breakdown in which the pathway is largely DPN-dependent and mitochondrial. This synthesis occurs from acetyl-CoA units, and these could be produced in necrotic tissue from the necrobiotic breakdown of fat, protein and carbohydrate. The increase of TPN- dependent isocitrate dehydrogenase activity is of special interest since i t has been shown that isocitrate is a necessary cofactor in fatty acid synthesis (Porter et al., 1957).

As there is no demonstrable increase of DPN diaphorase, succinate and DPN-dependent isocitrate dehydrogenase activity, the energy for this synthesis is presumably not derived from the tricarboxylic acid cycle, but it may be pro- vided by the hexose monophosphate shunt, as suggested by Langdon (1957). This pathway constitutes an alternative route for glucose-6-phosphate meta- bolism. Through the synthesis of pentose niicleotides it also provides a route for the building-up of nucleic acids (Scott and Cohen, 1951); methyl green-pyronin and gallocyanin stains, however, failed to show any increase of riboniicleic acid in the cytoplasm of TPN-diaphorase-positive macrophages, which makes i t unlikely that any increase in the activity of the pentose cycle is indicative of increased nucleoprotein synthesis.

The significance of increased alpha-glycerophosphate dehydrogenase activity, observed to be equally strong in the macrophages associated with necrosis and in other reactive cells such as astrocytes, is far from clear. The non-nucleotide- dependent, mitochondria1 form of the enzyme is considered to be linked to a phosphorylating electron-transport chain, whereas its non-particulate DPN- linked form provides a pathway to lipid synthesis. It has been suggested (Boxer

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FIG. 4.-Same lesion as in fig. 2. Intense deposition of formazan in reactive microglia (darkly staining). TPN- dependent isocitrate dehydrogenase. x 50.

FIG. 5 . - Necrotic lesion in spleen. Deposits of formazan within macrophages. TPN- dependent isocitrate dehydro- genase. x 420.

FIG. 6.-Same lesion as in fig. 2. Increased formazan deposition outlining leptomeninges and blood vessels only, virtually absent in microglia. DPN diaphorase. x 50.

ENZYME ACTI VIT k' I N MICROGLIA AA'D MACROPHAGES 573

and Shonk, 1960) that one role of the mitochondria1 form is to accomplish the transfer of hydrogen from the oxidation of glyceraldehyde-3-phosphate from the soluble phase of the cell to its insoluble, intramitochondrial compartment. In the present state of knowledge, however, one can only speculate that the demon- stration of apparent increased activity of this enzyme is a general indication of increased cellular metabolism.

I n conclusion it is felt that the demonstration of TPN diaphorase and TPN- dependent dehydrogenase activity in the fat-carrying cells associated with necrosis may indicate that they are engaged in fat synthesis; further biochemical studies are required to elucidate this point.

SUMMARY

A study by the tetrazolium salt technique of the oxidative enzyme activity of reactive microglia and somatic macrophages shows that these cells possess strong TPN diaphorase and TPN-dependent dehydrogenase activity, and weak or absent DPN diaphorase and DPN-dependent dehydrogenase act,ivity ; it is suggested they may be able to synthesise fatty acids from tissue breakdown products.

We are grateful to Dr L. W. Wattenberg, Department of Pathology, University of Minnesota, for his generosity in supplying one of us (L. J. R.) with the cofactors necessary for this work.

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