Acta Neuropathol (1992) 84:234 - 237 NuA mpathologia (~) Springer-Verlag 1992

Neuronal uptake of plasma proteins in brain contusions An immunohistochemical study

E. M. Loberg and A. Torvik

Division of Neuropathology, Department of Pathology, Ullev51 University Hospital, N-0407 Oslo 4, Norway

Received November 11, 1991/Revised, accepted March 2, 1992

Summary. Twenty- f ive cases of c e r e b r a l con tus ions o f va r ions age were e x a m i n e d i m m u n o h i s t o c h e m i c a l l y for n e u r o n a l u p t a k e o f a l b u m i n and f i b r i n o g e n . T h e n e u r o n s in t he d a m a g e d a reas we re heav i l y s t a i n e d in all cases , even in t hose of on ly a few m i n u t e s ' surv iva l , and t h e y r e m a i n e d pos i t i ve for s e r u m p r o t e i n s un t i l t h e y d i sap- p e a r e d f r o m the les ions . I n h e m a t o x y l i n a n d eos in- s t a i n e d sec t ions , n e u r o n a l changes were o b s e r v e d f r o m the first m i n u t e s a f t e r t he les ion b u t t h e y were ind i s t in - gu i shab le f r o m the s h r u n k e n " d a r k " n e u r o n s t ha t occu r as a r t i fac t s in p o o r l y f ixed m a t e r i a l . H o w e v e r , in c o n t r a s t to t he a r t i f ic ia l ly c h a n g e d cells , t h e t rue ly d a m a g e d ones t o o k up s e r u m p ro t e in s . I t is c o n c l u d e d t h a t s t a in ing wi th an t i s e r a aga ins t s e r u m p r o t e i n s m a y se rve as ea r ly m a r k e r s for n e u r o n a l in ju ry b e f o r e r e l i ab l e h i s to log ica l changes have d e v e l o p e d .

Key words: C e r e b r a l con tus ions - P l a s m a p r o t e i n s - I m m u n o h i s t o c h e m i s t r y

: . I m m u n o h i s t o c h e m i c a l o b s e r v a t i o n s on c r y o g e n i c b r a i n les ions have shown tha t d a m a g e d n e u r o n s t a k e up 6 e x t r a v a s a t e d p l a s m a p r o t e i n s wi th in a few m i n u t e s a f t e r 7

8 t he l e s ion [7], w h e r e a s n o r m a l co r t i ca l n e u r o n s r e m a i n 9 u n s t a i n e d b o t h in an ima l s and in h u m a n a u t o p s y m a t e - 10 r ia l du r ing the first 1 -2 days a f t e r d e a t h [8]. P r e l i m i n a r y i t o b s e r v a t i o n s o f b r a i n con tus ions also s h o w e d se lec t ive 12 u p t a k e o f p l a s m a p r o t e i n s in d a m a g e d n e u r o n s . T h e 13 p r e s e n t s tudy was p e r f o r m e d to see h o w soon a f t e r t h e 14 in f l i c t ion o f t h e les ion such u p t a k e t a k e s p lace . A s will 15 b e s een , n e u r o n a l u p t a k e o c c u r r e d w i th in a few m i n u t e s 16 17 a f t e r t h e d a m a g e a n d b e f o r e u n e q u i v o c a l h i s to log ica l 18 changes were d e m o n s t r a b l e . Th is s t u d y is p a r t o f a 19 s y s t ema t i c i nves t i ga t i on o f n e u r o n a l u p t a k e o f p l a s m a 20 p r o t e i n s a f t e r va r ious t ypes o f b r a i n les ions . 21

22 23 24 25

Correspondence to: A. Torvik (address see above)

Materials and methods

The material consisted of 25 cases of cortical brain contusions of known age. Most of the cases were victims of traffic accidents; a few had suffered other types of trauma. The length of survival varied from a few minutes to 15 days. The intervals between the time of death and autopsy varied from 8 h to 2 days (Table 1). Cases with anoxic cerebral damage or other types of brain complications were not included in the material.

The brains were fixed in 10 % phosphate-buffered formalde- hyde for 2 or 3 weeks. Blocks from the cortical contusions were then embedded in paraffin. Hematoxylin and eosin (H&E) was used as a routine stain. Immunostaining was performed with

Table 1. Main data for the 25 cases with brain contusions arranged according to increasing length of survival

Case no. Age Sex Survival time Post-mortem interval (h)

32 F Few min 40 29 M Few min 32 39 M Few min 12 12 M < 30 min 40 16 M < 45 min 28 31 F < 1 h 16 20 M 1 h 24 35 M 1-2 h 24 43 F 3 h 24 24 M 3 h 10 84 M 4-5 h 16 77 F 6 h 30 26 M 6 h 24 14 M 7-8 h 15 23 M 8-9 h 8

7 F 10-11 h 34 58 M 24 h 44 69 M 33 h 30 37 M 34 h 20 17 M 8 d 19 22 M 8 d 24 35 F 9 d 33 66 F 10-11 d 17 81 M 12 d 48 59 M 15 d 16

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Fig. 1. Dark, shrunken triangular neurons few minutes after the injury. Normal neurons marked with a r r o w s . H&E, x 250

Fig. 2. Dark, shrunken elongated neurons with wavy apical den- drites. Less than I h survival. H&E, x 250

Fig. 3. Dis tended neurons in the cortex with empty-looking cytoplasm and preserved nuclei less than 1 h after the injury. H&E, x 250

Fig. 4. Necrotic neurons with eosinophilic cytoplasm.Thirty-three hours ' survival. H&E, x 250

Fig. 5. Immunosta ined shrunken triangular neurons (type 1) and unstained distended neurons with empty-looking cytoplasm. Less than 1 h survival. Anti-f ibrinogen, x 250'

Fig. 6. Immunosta ined shrunken elongated neurons. Less than 1 h survival. Ant i -a lbumin, x 250

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antisera against human albumin and fibrinogen (Dakopatts A/S, Glostrup, Denmark).The antigen-antibody reaction was visualized with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method [9] using fast red as the cbromogen.

perivascular and albumin was somewhat more widely spread than fibrinogen. After longer intervals, the leakage was more widepread and there was no clear difference between the distribution of albumin and fibrinogen.

Results

General histology

Cerebral cortical hemorrhages were present in the lesions in all cases including those with only few minutes' survival. Otherwise, the early lesions were poorly delimited. After 12-24 h the necrotic tissue became demarcated from the normal cortex by a narrow zone of spongy tissue. Phagocytosis of the necrotic tissue by macrophages started at the edge of the lesions from the 3rd day and reactive astrocytes with eosinophilic cyto- plasm were visible from the end of the 1st week.

Neuronal changes. All cases that survived from minutes to 1 or 2 h showed two types of neuronal changes in H&E-stained sections. The first type (type 1) consisted of dark shrunken elongated or triangular neurons with small dark nuclei, and often with wavy apical dendrites (Figs. 1, 2). They consistently occurred in large numbers within the damaged tissue and rarely in the adjacent cortex. They were histologically indistinguishable from the "dark" neurons that occur as artifacts in immersion- fixed brains [3]. However, in contrast to the artificially changed ceils they stained positive with anti-albumin and anti-fibrinogen antibodies. Furthermore, they turned into eosinophilic shrunken cells during the first few hours after the trauma. These observations thus show that the dark neurons in recent contusions at least in part are truely damaged cells.

The second type of early change (type 2) consisted of distended neurons with empty-looking cytoplasm and lightly stained nuclei with preserved nucleoli and nuclear membranes (Fig. 3). The ultimate fate of these cells could not be determined. Similar cells, although less distended, could occasionally be seen also in the normal cortex.

In cases that survived from 1 to 6 h, an increasing number of the dark neurons turned into cells with eosinophilic cytoplasm and nuclei. Some of them were shrunken and elongated like the dark neurons described above, others were triangular resembling eosinophilic neurons following anoxic damage. From 12 to 24 h the cell and nuclear membranes of the eosinophilic neurons became increasingly blurred and the cells gradually disintegrated into poorly delimited eosinophilic ghost cells.

Immunohistochemical staining

Leakage of plasma proteins into the brain tissue could be demonstrated by immunostaining in all cases from the first few minutes after the trauma. In cases with the shortest survival periods, the staining was distinctly

Neurons. All cases, including those with the shortest survival periods, showed neurons that were positive for albumin and fibrinogen (Figs. 3, 4). In cases that sur- vived less than 1 h, nearly all positive cells were dark shrunken neurons of type 1 described above. They remained positive also when they turned eosinophilic after longer survival periods. All these eosinophilic neurons remained positive for serum proteins until they disappeared. In the cases with the longest survival periods, the ghost cells without visible nucleus were also positive. Practically none of the distended empty- looking neurons (type 2) were stained (Fig. 3).

Glial cells and macrophages. Positive glial cells were seen in all cases in the white matter, similar to normal cases [8]. Anti-albumin stained more glial cells than anti-fibrinogen. During the first few hours after the lesion, numerous glial cells with markedly distended cytoplasm were seen in the white matter adjacent to the damaged cortex. They were probably oligodendrocytes, and they were positive with anti-albumin but not with anti-fibrinogen. The macrophages were mostly negative; only scattered positive cells were seen. Reactive astrocytes were seen in the cases with the longest survival periods. They stained positive for albumin and fibrinogen.

Discussion

One of the most striking findings in the present study was the demonstration of truely damaged neurons within a few minutes after the trauma. They could not be distinguished from artificially changed cells in immer- sion-fixed material [3]. However, in contrast to artificial- ly changed cells [8], they took up plasma proteins, and they turned into eosinophilic cells after 1 h. "Dark" neurons may, therefore, be irreversibly damaged cells but immunostains are necessary to distinguish them from artifacts in immersion-fixed material.

The distended neurons in the lesions (type 2) were of more uncertain significance. Similar cells, although less distended, could occasionally be seen in normal brains and the fact that they were negative in immunostains suggests that they were not irreversibly damaged cells.

The early histological changes after traumatic lesions have previously received little attention. Mostly the neurons are described as "cells undergoing ischemic change" [1]. According to Bullock et al. [2], these changes occurred as late as 24 h after the lesion and the degeneration was, therefore, interpreted as a secondary or delayed process. Hekmatpanah and Hekmatpanah [4] also suggested that the shrunken neurons seen after trauma were caused by ischemic damage. In our mate-

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rial, the first histological changes were seen within a few minutes after the lesion. A direct traumatic mechanism, therefore, seems more likely than ischemic damage.

Lindenberg and Freytag [5] described two types of cell changes in thionin-stained material similar to those described here. They considered the distended type of cell as an artifact and the shrunken cells as truely damaged. The concept of shrunken cells as artifacts was not appreciated at that t ime and the significance of their findings has, therefore, been uncertain. A more recent experimental study described similarly shrunken cells 1 h after the injury in perfusion-fixed material [4] but it was questionable whether the traumatic lesion was adequately perfused. Thus, the sequential analysis and the immunohistochemical findings in the present study are necessary to prove that these early appearing shrunken cells are truely damaged.

The immunohistochemical findings in the present study resembled those seen after cold injuries [7]. Both types of lesions cause an immediate leakage of plasma proteins and uptake of proteins by the damaged neurons within a few minutes. Similar findings have been described previously in experimental studies after trau- matic lesions [6~ 11, 12]. However, the at tention has so far been directed more towards the vascular leakage than the neuronal uptake.

The mechanism of the rapid neuronal uptake is unknown. Membrane disruption is a posibility [4] but the intraneuronal concentrat ion rapidly becomes much higher than that in the surroundings and the affinity for protein must, therefore, be much higher in intracellular structures than in the surroundings.

Povlishock et al. [10] found indications that proteins may be taken up by reversibly damaged neurons after experimental concussions. All neurons in the present study were probably permanent ly damaged but the possibility that neurons may take up proteins after reversible lesions may be important in other types of neuronal damage.

In a previous study we demonstrated that normal neurons in the cortex are practically unstained for plasma proteins when examined within 1 or 2 days after death [8]. However, normal brain stem neurons did stain after short post-mortem intervals both in human cases and in immersion-fixed animal brains [8]. The present method for demonstrat ion of early neuronal damage,

therefore, is unsuitable for use in suspected brain stem lesions.

In conclusion, we have shown that plasma proteins are taken up into damaged neurons within a few minutes of the injury. The findings confirm that the vague histological changes that occur within minutes after the trauma indicate real damage, and the method can thus be used as a marker for early neuronal injury in human post mor tem material.

Acknowledgement. The authors wish to thank Ingeborg LCstegaard Goverud for excellent technical assistance.

References

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