Ada Nmropathc~l~ica

Acta neuropath. (Berl.) 41,207-210 (1978)

9 Springer-Verlag 1978

The Effect of Prolonged Experimental Hypereapnia on the Brain F r a n k M a t a k a s , J o s e f Birk!e, a n d J o r g e C e r v 6 s - N a v a r r o Department of Neuropathology, Klinikum Steglitz, Freie Universitfit Berlin, Hindenburgdamm 30, D-1000 Berlin 45

Summary. T h i r t e e n a d u l t r a b b i t s were e x p o s e d to a b r e a t h i n g air m i x t u r e c o n t a i n i n g a n increasing a m o u n t o f CO2 for eight weeks. W h e n the CO2 c o n t e n t r e a c h e d 9 Vol % the a n i m a l s b e c a m e a p a t h i c a n d lost b o d y weight. T h e E E G s h o w e d a r e d u c t i o n o f the a m p l i t u d e s o f 10 H z frequences. B l o o d gases revealed a n increase o f b i c a r b o n a t e b u t n o c h a n g e o f p H . T h e b l o o d b r a i n b a r r i e r w h i c h was tested w h e n the a n i m a l s were killed was n o t d i s t u r b e d . E n z y m e h i s t o c h e m i s t r y , light a n d e l e c t r o n m i c r o s c o p y r e v e a l e d t h a t m o d e r a t e b r a i n e d e m a h a d occurred. F r o m these results it is c o n c l u d e d that c h r o n i c h y p e r c a p n i a has a h y p n o t i c effect which in c o m b i n a t i o n with c h r o n i c e d e m a m a y depress vital activities c o n s i d e r a b l y . H o w e v e r , there seem to be n o irreversible m o r p h o l o g i c a l a l t e r a t i o n s o f the brain. Key words: B r a i n - H y p e r c a p n i a - E l e c t r o n m i c r o s copy - Histochemistry

C a r b o n d i o x i d e has a s t r o n g a n d c o m p l e x effect on the b r a i n function. D u r i n g a c u t e h y p e r c a p n i a the E E G d i s p l a y s a v a r y i n g a m p l i t u d e ( S w a n s o n et al., 1958; C u t l e r a n d B a r l o w , 1966) a n d m a y even b e c o m e isoelectric. T h i s h a s been s h o w n for m a n ( L a m i s s e et al., 1970) a n d e x p e r i m e n t a l animals. CO2 has also a m o s t p o w e r f u l effect o n c e r e b r a l b l o o d flow ( K e t y a n d S c h m i d t , 1948; Reivich, 1964). S o m e a u t h o r s have described an effect o f CO2 on the b l o o d b r a i n b a r r i e r ( C l e m e d s o n et al., 1958; C u t l e r a n d B a r l o w , 1966) w h i c h seems specially i m p o r t a n t for c h r o n i c h y p e r c a p n i a as it m a y o c c u r in p u l m o n a r y insufficiency. In this case p e n e t r a t i o n o f d r u g s o u t o f the b l o o d i n t o the b r a i n p a r e n c h y m a m i g h t be e n h a n c e d ( G o l d b e r g et al., 1963). H o w e v e r , all the e x p e r i m e n t a l studies so far p u b l i s h e d are restricted to the effect o f acute h y p e r -

c a p n i a . The following s t u d y was u n d e r t a k e n to investigate the effect o f c h r o n i c h y p e r c a p n i a o n b r a i n f u n c t i o n a n d m o r p h o l o g y . T h e r e has c o m e n o s t u d y to o u r k n o w l e d g e which has d o n e this before.

Material and Methods 13 rabbits between 6 and 12 months old, weighing between 3000 and 5000 g were used for the experiments. The animals were kept in a closed box (200 x 100 x 100 cm) which was ventilated with a defined gas mixture consisting of N2, 02 and CO2. Oxygen was kept at a level of 20 voI ~. At the beginning of the experiments CO2 of the breathing air was increased every third day by 1 ~ beginning with 0 vol %. When it was 9 ~o it was maintained at this level until the end of the experiments. CO2 concentration of the air was controlled continuously. Oxygen was controlled only once a week. Temperature and moisture of the box was controlled and kept at a normal level of 22~C and 50 - 70 % respectively. The experiment was finished after 8 weeks. A control series of 6 animals was kept as described above except for the fact that the box was ventilated with a gas mixture of 20 % O2 and 80 ~ N2. Blood gases, serum potassium and sodium, hematokrit and the EEG of all animals were controlled every 2 - 3 weeks. Withdrawal of blood from an ear artery and the EEG were performed the animals being inside their box. Six hours before sacrificing the animals they received 20 ml 2 ~ Evans blue intravenously. The animals were decapitated without previous narcosis. A small piece of the parietal lobe of the brain was put into chilled 5 % glutaraldehyde for electron microscopy. Another tissue sample from the same area was quickly frozen in liquid isopentane for enzyme histochemistry. The following tissue enzymes were determined: Adenosine triphosphatase (Padykula and Herman, 1955), alkaline phosphatase (Gomori, 1952), acid phosphatase (Barka, 1960), unspecific esterase (Spannhoff, 1967), acetylthiocholine esterase (Gomori, 1952), DPNreductase (Novikoff et al., 1961), lactate dehydrogenase (Pearse, 1961). Tissue samples were also taken from the lung, heart, liver, kidneys, muscles, and peripheral nerves for light microscopical investigation.

Results T h e r a b b i t s subjected to CO2 s h o w e d a d e c r e a s e d m o t o r i c activity after 1 - 2 weeks. A t the end o f the e x p e r i m e n t s the a n i m a l s were e x t r e m e l y a p a t h i c . T h e

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Acta neuropath. (Berl.) 41 (I978)

Table 1. Parameters of hypercapnic (H) and control (C) animals. Hypercapnic animals differ from control animals only with respect to pCO2 tension and HCO2. All values are mean values Week 1

Week 2

Week 3

Week 8

H

C

H

C

H

C

H

C

CQ-concentration in box (vol %)

0

0

5

0

7

0

9

0

Blood (arterial) pH pCO2 (ram Hg) HCO3 (mVal/1) PO2 (ramHg) Hematokrit (%) Potassium (mM/1) Sodium (mM/1)

7.29 28.4 15 95 35 6.3 138

7.36 31 19 101 36 4.2 140

7.30 45.5 21 113 30 4.5 146

7.38 27 18 97 34 3.6 143

7.33 40.0 23 95 31 4.4 140

7.36 31 19 96 33 3.4 139

7.33 60 29 100 28 4.9 142

7.42 28 21 95 31 3.7 140

Table 2. Activitiesof some enzymesin various tissue elements of the parietal lobe. Enzymesand cell types not included in this table were not influenced by hypercapnia. H: animals exposed to CO2, C: control animals. - no activity; + moderate activity; + + strong activity

Enzyme

Neurones

Astrocytes Vessels

LDHase

C H

+

+ ++

++ ++

Acid phosphatase

C H

+ +

-

+"

Unspecific esterase

C

++

-

+~

H

+

--

++~

a

Activityin perivascular cell elements

ventilation frequency increased from about 50/min at the beginning to an average of 130/min when the CO2 rose to 3 vol %, but decreased to an average value of 60/min when the CO2 concentration was 9 vol %. The average body weight dropped f r o m a mean value of 3490 g at the beginning to 2825 g. CO2 tension in the arterial blood of the experimental animals averaged at 60 m m H g as soon as CO/ content of the box had reached the level of 9 vol %. Potassium, sodium, blood p H did not alter significantly from normal values (Table 1). Only bicarbonate increased from 1 5 29 mVal/1. Hematocrit was slightly decreased. The E E G of CO2-animals showed a slight decrease of frequency around 10 Hz. In contrast to the experimental animals the control animals showed no abnormalities of either behaviour, blood values or the E E G (Table 1). There were no significant histological alterations of the heart, lungs, kidneys, liver, muscles or peripheral nerves. The Evans blue had stained the organs of all animals blue. However, in none of the animals either experimental or control the brain tissue was colored. The tissue examined by enzyme histochemistry contained cortex and medulla of the parietal lobe.

Hypercapnia had increased the activity of lactate dehydrogenase in neurones and glial cells. The unspecific esterase and, to a minor degree, the acid phosphatase were increased little in perivascular cell elements of the hs)percapnic animals (Table 2). Hypercapnia had no effect on NADH-reductase, alcaline phosphatase, acetylthiocholine esterase, a n d ATPase. There was also no effect on diffuse tissue activity of any enzyme. Under the electron microscope astrocytes in experimental animals showed edematous swelling. Pericapillary astrocytic processes of the grey and white substance were most affected (Fig. 1). Their cytoplasm had an increased electron density and they contained only few cell organelles. In a few cases pericapillar swelling had led to a narrowing of the capillary lumen. There was hardly an astrocytic process of the neuropil that appeared normal although astrocytic swelling was less in the neuropil than perivascular (Fig. 2). In 6 animals astrocytes contained m a n y glycogen gram ules (Fig. 3). Astrocytic edema was not found in any of the control animals, except for a few processes which seemed to be a little enlarged. Capillaries appeared normal in all animals except for the fact that some were narrowed in hypercapnic animals by swollen astrocytic processes. There were m a n y dark nerve cells both in hypercapnic and control animals. Discussion

The physiological resporise of our animals to hypercapnia is in good agreement with earlier investigations. Breathing frequency is increased initially but may decrease in chronic states of hypercapnia (Geisler and Rost, 1972). Blood p H is not changed, but bicarbonate is elevated (Polak et al., 1961). The E E G showed a depression of 10 Hz amplitudes (Sieker et al., 1960). The behaviour of the animals can be attributed to the hypnotic effect of C O / ( W o o d b u r y and Karler, 1960).

F. Matakas et al. : The Effect of Prolonged Experimental Hypercapnia on the Brain

Fig. 1 Rabbit, 8 weeks hypercapnia. Parietal cortex. Capillary with two pericytes one of which contains lysosomes. The capillary wall appears normal. Severe pericapillar astrocytic edema, x 5395

Fig. 2 Rabbit, 8 weeks hypercapnia. Parietal subcortical area. Numerous swollen astrocytic processes. The neuropil appears normal otherwise. • 8550

Fig. 3 Rabbit, 8 weeks hypercapnia. Parietal cortex. Perivascular astrocytic process with numerous glycogen granules, x 4260

209

210

The depressive effect of CO2 on the central nervous system has also been studied in man and was named respiratory encephalopathy (Lamisse et al., 1970). The motoric inactivity of our experimental animals can well be ascribed to this syndrome. Clemedson et al. (1958) and Cutler and Barlow (1966) found an increased permeability of the blood brain barrier if the animals had been exposed to a gas mixture containing up to 30 vol % of CO2. The latter group did not find a leak of the blood brain barrier if the CO2 content was 10 vol % or less. In our experiments a gross impairment of the blood brain barrier was not detectable. In contrast to many physiological investigations morphological studies are only few and there are none so far which deal with the morphological sequelae of chronic hypercapnia. The results of our morphological investigations were surprising: We only found astrocytic swelling, an increase of LDHase in glial cells and an increase of catabolic enzymes in perivascular cells of the parietal region. The increase of LDHase activity can be regarded as a result of astrocytic swelling (Adams, 1965; Rasmussen and Klatzo, 1969). Thus the only significant effect of severe chronic hypercapnia was moderate brain edema. It must be considered, however, that the pattern for various enzymes is not equal in all regions of the brain (Anderson et al., 1962; Friede, 1962). We can thus not exclude that in some other areas than those examined tissue enzymes were ~tffected by hypercapnia.Enzyme histochemistry, moreover, is a rather gross method for the examination of metabolic effects. It might be assumed also that the astrocytic swelling observed by electron microcopy was merely an artifact caused by the preparation of the tissue. However, there a good reasons against this assumption. Remarkable astrocytic swellirig was not observed in control animals. This indicates that tissue preparation was optimal. Perivascular astrocytes had not a watery appearance but contained an electron dense material or even glycogen. We car(therefore assume that astrocytic swelling was an intravital and not a postmortem phenomenon. Respiratory encephalopathy is according to Lamisse et al. a disease which affects central nervous function. There are unspecific psychiatric and neurological symptoms, which are quickly reversible if respiration becomes normal. The substrate of these symptoms seems to be the hypnotic effect of CO2 combined with brain edema. Changes in cerebral blood flow may play a role in acute hypercapnia but do not occur in chronic hypercapnia (Betz, 1965). A disturbance of the blood brain barrier requires rather excessive CO2 concentrations which are hardly observed in human pathology.

Acta neuropath. (Berl.) 41 (1978)

"Acknowledgements. We gratefully appreciate the excellent technical assistance of Mrs. G. Bender, Mrs. B. Strehler and Mrs. G. Kluge.

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Received July 5, 1977/Accepted December 5, 1977