Journal of Neurology, Neurosurgery, and Psychiatry, 1973, 36, 1046-1052

Histochemistry of enzyme response to trauma in theneocortex and corpus callosum of developing

rat brainN. ROBINSON1

From the Department of Anatomy, The London Hospital Medical College, London

SUMMARY The enzyme response to injury of the brain was well localized and limited. Someenzymes, even in 12 day old brain, increased rapidly, mainly in neocortical glial cells. In the corpuscallosum enzymes were not significantly hyperactive before the light myelination stage. Somehyperactivity declined after 21 days. Oxidative processes and phosphate metabolism were mostdisturbed.

The effect of intracerebral penetration of asyringe needle on the metabolism of nerve andglial cells in the adult rat brain is reflected inwide variations in the response of enzyme activi-ties to trauma, the variations persisting in theneocortex and subadjacent white matter over aperiod of 31 days post-injection (Robinson,1969a).Driving a needle through the neocortex and

into the subadjacent white matter results intissue necrosis in the line of needle penetrationwith diminishing effects receding from the site ofnecrosis. Morphological, colloidal, and chemicalchanges transmitted through the tissue by theneedle produce disturbances of the metabolismof nerve cells, glial cells, and myelinating fibresand it is thought that the magnitude of thedisturbances and the rates of recovery would bedifferent at different stages of development. Thispaper describes the changes in activities ofenzymes of important metabolic pathways ofbrain at the site of intracerebral penetrationinto the neocortex and corpus callosum atdifferent stages of myelination.

METHODS

Male Sprague-Dawley rats, aged 12, 20, and 30 daysold, were used. The animals were lightly anaesthet-ized, hair shaved off the scalp, and a sagittal incision1 Address for reprints: Department of Anatomy, The LondonHospital Medical College, London E. 1. Z.A. I.

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made under aseptic conditions to expose the skull.A burr-hole was drilled in the skull to expose thedura mater at area 4 of Krieg (1946) and a sterilizedLuer 27G needle introduced to a predetermineddepth to penetrate the neocortex and corpus callo-sum. The needle was withdrawn and 300,000 i.u.benzathine penicillin was administered intraperi-toneally before suturing the incision; animals killedone day after injection were not sutured.The animals were killed at intervals of 1, 2, 4, 8,

14, 21, and 31 days after operation. The brains wererapidly dissected, instantly frozen and fresh frozensections 10 ,um thick were cut on a cryostat. Theenzymes examined by techniques previously de-scribed (Robinson, 1969b) were as follows: NADH2-diaphorase, glucose 6-phosphate (G6-PDH), lactate(LDH), succinate (SDH) and a-glycerophosphate(ot-GPDH) dehydrogenases, acid phosphatase, Mg2 I-ATPase, 5'-nucleotidase, thiamine pyrophosphatase(TPPase), monoamine oxidase (MAO), acetyl-cholinesterase (AChE) and non-specific esterase(NSE).

RESULTS

CLINICAL SIGNS All the animals survived theoperation and showed no apparent ill-effects orabnormalities in behaviour immediately after theoperation or over the period of recovery.

12 DAY OLD BRAIN Onset of myelination ofcorpus callosum Up to four days post-injectiona line of tissue necrosis produced by penetrationof the needle was seen, but at eight days the

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lesion was identified only by changes in enzymeactivity.At one day post-injection a narrow well-

defined region of neocortex showed weakNADH2-diaphorase, LDH, and G6-PDH reac-tions in the cytoplasm of glial cells and LDH wasslightly increased in neuropil; oxidative enzymereactions in the corpus callosum were negative.Acid phosphatase showed weak activity innerve cells, along fragmented axons, and withina few macrophages in the neocortex but noresponse was apparent in the corpus callosum.In the neocortex ATPase was slightly increased inswollen glial cells but the corpus callosum showed

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no response. Only a line of isolated scatteredsingle cells showed a raised TPPase activity inthe neocortex and corpus callosum. NSE activitywas raised in nerve and glial cells but the localiza-tion was poor. No changes in MAO or AChEactivities were detected.By four days post-injection a raised NADH2-

diaphorase activity was seen in nerve cells, inglial cells, and neuropil adjacent to tissuenecrosis. Numerous glial cells in the neocortexbut only scattered cells in the corpus callosumexhibited raised acid phosphatase activity (Fig.1). A poorly localized moderately increasedATPase activity was seen in scattered glial cells

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FIG. 2. Twelve day old rat; four days post-injection.Well-defined raised 5'-nucleotidase activity in swollengial cells of neocortex (above) and diffuse activity incorpus callosum (below). x 310.

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in the neocortex but the cytoplasm of some nerveand glial cells receding away from the area ofmost activity also showed a weak response.Swollen glial cells showed prominent raised 5'-nucleotidase activity but nerve cells showed asimilar reaction to that seen at two days. Themost marked change at four days was in thecorpus callosum where a poorly localized reac-tion, but with recognizable glial cells, showedincreases in 5'-nucleotidase; some fibres alsoshowed the enzyme reaction, albeit weaker, notnormally seen (Fig. 2). TTPase in nerve cellsalong the lesion had increased slightly but otherstructures showed no distinct response (Fig. 3).

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FIG. 3. Twelve day old rat; four days post-injection.Sparse nerve cells (arrows) with slightly raised thi-amine pyrophosphatase activity. x 125.

Other enzymes showed no marked changesexcept a slight increase in MAO activity in somenerve cells in the neocortex (Fig. 4).By eight days, only raised enzyme activities

enabled the lesion to be identified, since foci ofdamaged tissue were seldom seen. In the neo-cortex a more intense ATPase reaction was seenin glial cells and in some axons in the neocortexand also more numerous astrocytes with raised5'-nucleotidase and TTPase activities. No signifi-cant changes in the corpus callosum were ob-served.

20 DAY OLD BRAIN Light myelination of coipus

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FIG 4 Twelve day old rat; four days post-in]ection.Slightly raised MAO activity in a few nerve cells(arrows) similar to Fig. 3. x 125.

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callosum The response of oxidative enzymes todisturbance was more marked than in 12 day oldanimals. The reactions of these enzymes in theneocortex one day post-injection were generallystill weak but the localization was more distinct;more glial cells showed increased oxidativeenzyme activity, particularly NADH2-diaphorase(Fig. 5), but the neuropil remained unchanged.In the corpus callosum swollen glial cells withmainly NADH2-diaphorase, LDH, and a-GPDH activities were seen for the first time atone day post-injection; some oligodendrocytesalso showed a reaction but there was no responsein nerve fibres. The reactions of phosphatases inl W 4 l, . 1. .. X . . W.,) ...

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FIG. 5. Twenty day old rat; one day post-injection.Strong NA DH2-diaphorase activity mainly confined toglial cells in the neocortex. x 310

unaffected neocortex were generally more in-tense but the activities in response to injury wereonly slightly raised, comparable with the 12 dayold brain. In the corpus callosum a marked acidphosphatase responise in swollen glial cells andnerve fibres was seen and 5'-nucleotidase showeda prominent glial cell reaction at one day post-injection. Other enzymes of phosphate metabol-ism showed little change except within a fewscattered cells. A raised NSE activity in affectednerve cells was more precisely localized than in12 day old brain and several prominent glialcells exhibited raised activities in the corpuscallosum. Other enzymes, particularly NSE,

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FIG. 6. Twenty day old rat;o. e day post-injection.Moderately raised LDH activity in neocortex andslightly raised activity in oligodendrocytes and nervefibres of corpus callosum. x 125.

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MAO, and AChE, did not show any significantincreases in activity up to 14 days post-injection.At 21 days post-injection the most significantchange was a decrease in number of cells withraised enzyme activities.

30 DAY OLD BRAIN Substantial myelination ofcorpus callosum More rapid increases in en-zyme activities were observed in both neocortexand corpus callosum in response to injury at thisage than in younger animals; the lesion alsoappeared more extensive than before.At one day post-injection NADH2-diaphorase

activity was stronger in many more swollen glial

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cells of the neocortex than in younger animalsand the strong reaction in neuropil frequentlymasked the response in nerve cells. The reactionof LDH to injury was less intense than NADH2-diaphorase in the neocortex; in the corpuscallosum a well-localized slightly raised LDHactivity was seen in lines of oligodendrocytes andnerve fibres (Fig. 6). In the neocortex and corpuscallosum acid phosphatase activity was ab-normally strong within glial cells and manyfibres (Fig. 7). In the corpus callosum raised acidphosphatase activity was seen some length along

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FIG. 7. Thirty day old rat; one day post-injection.

Abnormally intense acid phosphatase activity in glialcells of neocortex and glial cells and nerve fibres incorpus callosum. x 50.

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FIG. 8. Thirty day old rat; one day post-injection.Intense acid phosphatase activity (normally absent) inswollen glia and nerve fibres adjacent to injury in cor-pus callosum (left hand side). Poorly localized activityin nerve cells at top right hand side. Arrow denotesneedle penetration. x 310.

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nerve fibres receding from the lesion accom-panied by hyperactive glial cells (Fig. 8). ATPaseand 5'-nucleotidase reactions were similar tothose in the 20 day old rats, while TPPase, NSE,MAO, and AChE showed no distinguishingreactions. At four days post-injection the dis-tribution and raised intensity of G6-PDH in theneocortex was similar to other oxidative enzymesbut in the corpus callosum a raised G6-PDHactivity was mainly within glial cells close toinjury (Fig. 9) and not along nerve fibres as forLDH in Fig. 6 and acid phosphatase in Fig. 7.Raised ATPase activity was seen in few glialcells and was negligible in neuropil and in nervefibres in contrast with acid phosphatase.

FIG. 9. Thirty day old rat; four days post-injection.Raised glucose 6-phosphate dehydrogenase activity inglial cells of neocortex and corpus callosum with no

response in nervefibres (compare LDH in Fig. 6). x 50.

At subsequent times the corpus callosumenzymes showed further increases, particularlyNADH2-diaphorase, LDH, and acid phospha-tase; ATPase and acid phosphatase showedmore distinct raised activities along nerve fibres,some appearing enlarged with strong reactionswhich were normally absent. TTPase showed anunusual well-localized raised activity in a fewscattered swollen glial cells at 21 days post-injection. In the neocortex MAO was localizedalong the lesion as lines of beads, suggestingaccumulation of the enzyme within smallglobules. By 21 days post-injection there wereresidual areas of tissue necrosis and the appear-ance of scarring was more in evidence in neo-cortex than in corpus callosum. Raised activityof phosphatases had diminished in cells but bareaxons still exhibited a reaction.

DISCUSSION

In experiments on intracerebral injection, syringeneedle or electrodes smaller than the Luer 27Gare frequently employed but a substantial furrowinto the cerebrum was necessary to observerecovery in young animals before morphologicaland enzyme changes were masked by growth ofnew tissue. Damage to tissue along the site ofneedle penetration was surprisingly limited andthe response of enzyme activity in the neocortexwas invariably confined to cells of the peripheryof damaged tissue but in the corpus callosum thelesion was usually more extensive and the enzymeresponse was frequently more widely distributed.

In the corpus callosum the 20 day old ratshowed the first distinct oxidative enzyme reac-tion along nerve fibres where lines of reactiveoligodendrocytes receded from the site of injurysimilar to a reaction reported at the periphery ofplaques of multiple sclerosis (Ibrahim andAdams, 1963; Friede and Knoller, 1964). How-ever, the responses of the oxidative enzymeswere dissimilar; LDH activity was less intensein the corpus callosum than in neocortex, G6-PDH activity was equally intense in both regions,while a-GPDH activity was also equally intensebut within many fewer glial cells in the corpuscallosum compared with neocortex. The intenseacid phosphatase response in glial cells andmyelinated nerve fibres also observed in otherpathological states (Friede, 1962; Robinson,

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1969a) persisted up to 14 days post-inijection butthen diminished; the elevated ATPase and 5'-nucleotidase activities also diminished in intens-ity and distribution, but not before 21 days. 5'-nucleotidase was the only enzyme showing adistinct increase in activity in the early myelina-ting stage of the corpus callosum up to eightdays post-injection, although raised acid phos-phatase activity was apparent in scattered glialcells. After the initial appearance of this enzymeresponse, subsequent enzyme changes in the cor-pus callosum were less distinct, since the lesionwas difficult to locate and was usually seen as ascarring with fewer hyperactive swollen glialcells retaining strong enzyme activity. Only inthe more substantially myelinated corpus callo-sum was acid phosphatase seen to follow dam-aged fibres, in some instances as far medially asthe midline.

In the developing neocortex enzyme reactionsshowed closer patterns of response to injurythan seen at each stage of myelination in thecorpus callosum, where it has been suggestedthat breakdown products of myelin are possiblestimuli (Osterberg and Wattenberg, 1962). Inolder animals the number of glial cells showingenlargement and raised enzyme activity increasedup to three weeks but in the 12 day old rats mor-phology and enzyme changes were not detectabletwo weeks after injection, being masked by rapidovergrowth of new tissue. During developmentthe enzyme changes in nerve cells were generallysmall but those cells at the periphery of neo-cortical damage showed a greater increase inenzyme activity than cells more distant. Some-times this increase was masked by a strongneuropil reaction; the nerve cell populationshowing abnormal reactions, however, dimin-ished rapidly with the distance from injury.Detectable changes in MAO, NSE, AChE, andTPPase activity appeared to be more withinnerve cells than glia and confined to cells in theline of needle penetration.

CONCLUSION

The extent of tissue damage due to penetration

of a Luer 27G syringe needle was surprisinglylimited and the enzyme response, which waswell localized, was generally confined to theperiphery of the injury. In the neocortex theresponse was rapid and intense for enzymes ofoxidative processes and phosphate metabolism,even at the onset of myelination stage. In thecorpus callosum enzyme response in the samemetabolic pathways was not significant untillight myelination when the response was rapid.As the corpus callosum became more myelinated,so the intensity of enzyme response to the lesionincreased. The hyperactivity of some enzymesappeared to diminish 21 days after injury andevidence of some scarring was first seen; otherenzymes, particularly thiamine pyrophosphatase,MAO, AChE, and non-specific esterase, showedno significant response to injury in the neo-cortex or corpus callosum at any stage of thedeveloping brain.

The Friedreich's Ataxia Group and the Action forthe Crippled Child Fund are thanked for financialassistance.

REFERENCES

Friede, R. L. (1962). An enzyme histochemical study ofcerebral arteriosclerosis. (With some data on the patho-genesis of periarterial scars.) Acta Neuropathologica, 2,58-72.

Friede, R. L., and Knoller, M. (1964). Quantitative enzymeprofiles of plaques of multiple sclerosis. Experientia, 20,130-1 32.

Ibrahim, M. Z. M., and Adams, C. W. M. (1963). Therelationship between enzyme activity and neuroglia inplaques of multiple sclerosis. Journal of Neurology, Neuro-surgery, and Psychiatry, 26, 101-1 10.

Krieg, W. J. S. (1946). Accurate placement of minute lesionsin the brain of the albino rat. Quarterly Bulletin of theNorthwestern University Medical School, 20, 199-208.

Osterberg, K. A., and Wattenberg, L. W. (1962). Inductivefactors in gliosis. Proceedings of the Society of Biology andMedicine, 111, 452-455.

Robinson, N. (1969a). Histochemical changes in neocortexand corpus callosum after intracranial injection. Journal ofNeurology, Neurosurgery, and Psychiatry, 32, 317-323.

Robinson, N. (1969b). Histochemistry of human cervicalposterior root ganglion cells and a comparison withanterior horn cells. Journal of Anatomy, 104, 55-64.

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