Acute ethanol intoxication can inhibit leucocyte mobilization in the corneal wounds

Exp. Eye Res. (1981) 33, 631 639

A c u t e E t h a n o l I n t o x i c a t i o n c a n Inhibit L e u c o c y t e M o b i l i z a t i o n in t h e C o r n e a l W o u n d s * M. A V A I ~ I A t , P. K . B A S U AND B. K A P U R

Department of Ophthalmology, University of Toronto and Addiction Research Foundation, Toronto, Canada (Received 11 November 1980 and accepted 24 March 1981, New York) The susceptibility to various kinds of infection in alcoholics has been attributed to the impairment of leucocyte functions by ethanol. As the possibility of corneal injury is ever present in the intoxicated person we wished to study the pattern of leucocyte mobilization into wounds of the cornea (an avascular tissue) of rabbits intoxicated with alcohol. Thirty-six rabbits were divided into two equal groups. One group (alcohol group) was injected intravenously with 25 % ethanol (20 ml/kg) to produce intoxication. The other group (control group) did not receive the agent and was anaesthetized with intravenous pentobarbital sodium. Identical non-penetrating central and peripheral trephine incisions (4 mm in diameter and 0'1 mm in depth) were made in both corneas of each rabbit. Two, 4 or 6 hr following the injury, cellular samples were obtained by pressing a cover slip on the surface of the lesions (the impression technique). The animal was then killed for corneal histology. Leucocyte (PMN) counts were made on samples obtained by both the impression and histological techniques. In the alcohol group, the average blood alcohol level in the animals at the time of killing was about 400 mg/100 ml. At each of the three time periods, during which the cornea remained avascular, the alcohol group showed a significantly lower number of PMNs in the corneal lesions (both central and peripheral) as compared to the corresponding lesions in the control group (P < 0"1~)'001). The total and differential blood leucocyte counts in both groups were similar. Our results showed that alcohol inhibits mobilization of PMNs in corneal wounds at least for 6 hr following injury. Key words:acute ethanol intoxication ; polymorphonuclear cells ; leucocyte migration; corneal wounds ; rabbit.

I. I n t r o d u c t i o n As c h r o n i c alcoholics t e n d to s h o w a g r e a t s u s c e p t i b i l i t y to v a r i o u s k i n d s o f i n f e c t i o n , e t h a n o l ( e t h y l alcohol) has b e e n s u s p e c t e d t o be one o f t h e a g e n t s w h i c h m i g h t m o d i f y i n f l a m m a t i o n b y a l t e r i n g l e u c o c y t e f u n c t i o n s ( M a c G r e g o r , G l u c k m a n a n d Senior, 1978). N o r m a l l y , w o u n d s in tissues w h i c h h a v e a d i r e c t b l o o d s u p p l y are i n f i l t r a t e d b y P M N s w i t h i n a few m i n u t e s a f t e r t r a u m a ( S n y d e r m a n a n d M e r g e n h a g e n , 1972). B u t in a n i m a l s a n d m a n f o l l o w i n g e t h a n o l a d m i n i s t r a t i o n , t h e a c c u m u l a t i o n o f P M N s a t t h e site o f i n d u c e d i n f e c t i o n or t r a u m a (e.g. skin, lungs, p e r i t o n e u m , etc.) is d e c r e a s e d (Pickrell, 1938; L o u r i a , 1963; Moses, G e s c h i c k t e r a n d E b e r t , 1968; B r a y t o n , S t o k e s , S c h w a r t a n d L o u r i a , 1970; J o h n s o n , 1975). A l t h o u g h t h e r e is still some~ c o n t r o v e r s y , it is g e n e r a l l y a g r e e d t h a t g r a n u l o c y t e s m a i n t a i n n o r m a l c h e m o t a x i s , ~phagocytosis a n d i n t r a c e l l u l a r killing o f b a c t e r i a e v e n in s p i t e o f a h i g h c o n c e n t r a t i o n o f a l c o h o l in t h e b l o o d ( J o h n s o n , 1975). T h e p e r i p h e r a l b l o o d l e u c o c y t e c o u n t s o f e t h a n o l t r e a t e d a n i m a l s (Louria, 1963) or h u m a n (Moses e t al., 1968) do n o t differ f r o m t h a t o f t h e u n t r e a t e d controls. H o w e v e r , s u b l e t h a l c o n c e n t r a t i o n s o f a l c o h o l s i g n i f i c a n t l y i n h i b i t a d h e r e n c e o f t h e g r a n u l o c y t e s to t h e * Presented at the Fourth International Congress for Eye Research, New York, 1980. t Supported by a fellowship from the Canadian National Institute for the Blind out of the E. A. Baker Foundation for the Prevention of Blindness. Reprint requests to : Dr P. K. Basu, Department of Ophthalmology, University of Toronto, 1 Spadina Crescent, Toronto, Ontario M5S 2J5, Canada. 0014-4835/81/120631 +09 $01.00/0

9 1981 Academic Press Inc. (London) Limited 631

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v a s c u l a r endothelial surfaces, a p r o p e r t y which is essential for their diapedesis from blood circulation (Marchesi a n d Florey, 1960). However, e x a c t l y how the leucocyte diapedesis is affected b y e t h a n o l is n o t y e t fully known. Various factors have been i m p l i c a t e d in the i m p a i r m e n t of leucocyte mobilization under the influence of alcohol (Moses et al., 1968; T i s m a n a n d H e r b e r t , 1973; Spagnuolo a n d MacGregor, 1975; G l u c k m a n a n d MacGregor, 1978; Buckley, V e n t u r a a n d MacGregor, 1978). W h e t h e r the s t a t e of the v a s c u l a r e n d o t h e l i u m (Majno, Shea a n d Leventhal, 1969) is also altered under the influence of alcohol is also n o t known. As far as we could d e t e r m i n e the effect of acute e t h a n o l i n t o x i c a t i o n on the infiltration of corneal wounds b y leucocytes has n o t been investigated. As the possibility of corneal injuries is ever p r e s e n t in the i n t o x i c a t e d person, we wished to s t u d y the p a t t e r n of P M N mobilization into corneal wounds of h e a l t h y animals i n t o x i c a t e d with ethanol. I n our experiment, identical t r a u m a t i c lesions were p r o d u c e d in the cornea of two equal groups of animals. One group (alcohol group) received ethanol a n d the other group (control group) did not. The two groups were c o m p a r e d chronologically up to 6 hr (Issekutz a n d Movat, 1980) following the infliction of corneal i n j u r y b y e s t i m a t i n g the degree of P M N infiltration into the wounds. F o r cell counting two different m e t h o d s were used.

2. Materials and M e t h o d s Thirty-six albino, male, New Zealand rabbits weighing between 4 and 5 kg, with normal eyes, were divided into two equal groups: (1) the control group and (2) the alcohol group. The animals (n = 18) of the control group were anaesthetized intravenously with sodium pentobarbital (Nembutal, Abbott Laboratories Ltd., Montreal) with a dose of 30 mg/kg. The animals (n = 18) of the alcohol group were injected intravenously with 25 % ethanol in a dose of 20 ml/kg (600 mg of alcohol per l0 ml of blood). This made them stuporous. On each cornea of the 36 animals, one central and one peripheral non-penetrating circular incision (4 mm in diameter and 0"1 mm in depth) were made with a Castroviejo corneal trephine (Storz Instrument Co., St Louis, Mo.). The centre of the peripheral incision was placed 5 mm away from the limbus - the right being kept on the 10.30 o'clock meridian and the left on the 1.30 o'clock meridian. The injured portion of the cornea was kept covered by the eye lids. After 2, 4 or 6 hr following the injury, cellular samples were collected using the impression technique (Hershenfeld et al., 1981) from the traumatic lesions (using six animals per group for each period) by gently pressing a plastic coverslip (Thermanox (TM) Cat. no. 5408, Lot. no. 118135, Lux Scientific Corp., Newbury Park, California) over the entire trephine wound. The materials attached to the cover slip were air dried and stained with Diff-Quik (R) (Harleco, Gibbstown, N.J.). The polymorphonuclear leucocytes were counted under a microscope (fitted with a grid for counting purposesi in a total surface area of 1"00 mm 2 per cover slip from at least three randomly selected sites using 100 • magnification [Fig. l(a)]. Immediately after taking the cover slip samples, each animal was killed with an overdose of Nembutal (120-250 mg/kg). Both corneas with a scleral rim were excised from each rabbit, fixed with 10 % formalin, sectioned in paraffin and stained with G.iemsa stain. The number of PMNs in at least four different sections (50-100 #m apart) of each lesion, was counted in a total area of l'00 mm 2per section (including both sides of the corneal incision) under a • 450 magnification [Fig. l(c)-(f)]. Just before killing, blood samples were taken from the animals for counting the peripheral blood leucocytes (total and differential) and for estimating the blood alcohol levels in the alcohol group.

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FIG. 1. Rabbit cornea [(a) Impression technique, Diff-Quik(~); (b)-(f) Histology, Giemsa Stain]. (a) Cellular impression on a plastic cover slip [Thermanox (TM), x 22-5]. Insert shows cells at a hi_gher magnification. • 120. (b) The limbus (control group) showing a subconjunctival accumulation of leucocytes 2 hr following trauma, some of which emerging through the epithelial layer (arrow). • 90. (c) Central corneal wound of an animal of the control group, showing a leucocyte infiltration in the stroma 2 hr following the injury. • 90. (d) Central corneal wound of an animal of the alcohol group showing fewer leucocytes infiltrating the stroma, 2 hr following injury, x 90 (Compare c). (e) Central corneal wound of another animal of the control group showing a leucocyte infiltration in the stroma 2 hr following injury, x 360. (f) Central corneal wound of another animal of the alcohol group showing fewer leucocytes infiltrating the stroma 2 hr following the injury, x 360 (Compare e).

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3. Results W i t h r e g a r d to the P M N response to the corneal injury, the two eyes of each r a b b i t b e h a v e d similarly. The n u m b e r of P M N s in the two central or two p e r i p h e r a l lesions in each r a b b i t were v e r y close. I n each eye t h e n u m b e r of P M N s in the peripheral lesions was greater t h a n t h a t from the central lesions, irrespective of the group or sampling t i m e or sampling technique. I n b o t h groups, h a r d l y a n y P M N s were observed in the corneal tissue between the lesion and t h e limbus 2 hr following the injury. B u t after 2 hr, PMNs infiltrated the tissue intervening the i n j u r e d site and the limbus (Fig. 2), the n u m b e r of which progressively increased with time.

FIG. 2 Histology of a rabbit cornea with a peripheral wound, showing infiltration of leucocytes in the anterior stroma (4 hr sample) intervening the site of wound (W) and limbus (L), Giemsa, x 30. Insert shows cells at a higher magnification, x 160.

The pooled d a t a for the central or peripheral lesions from all animals in each group with respect to a given s a m p l i n g time (2, 4 or 6 hr) a n d a given sampling technique have been shown in Tables I (impression techniques) a n d I I (histology). I t will be seen in these tables t h a t in all instances the d a t a o b t a i n e d b y the impression a n d histological techniques r a n parallel. The n u m b e r of PMNs in the central lesions of the control group was g r e a t e r t h a n t h a t in the central lesions of the alcohol group. The n u m b e r of PMNs in the p e r i p h e r a l lesions of the two groups also v a r i e d in a similar fashion. Consequently, the t o t a l n u m b e r of P M N s in the corneal lesions of the control group was g r e a t e r t h a n t h a t of the alcohol group. Our results t h u s show t h a t alcohol i n t o x i c a t i o n can reduce the mobilization of P M N s into the corneal wounds a t least during t h e first 6 hr following t r a u m a to the tissue [Fig. 1 (c)-(f)]. There was no difference between the p e r i p h e r a l blood leucocyte counts between the two groups at a n y time. The average blood alcohol concentrations were 441"5, 399"0 a n d 379"5 m g / 1 0 0 ml a t 2, 4 a n d 6 hr respectively.

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4. Discussion The cornea being an avascular tissue, offers an excellent system for studying the kinetics of leucocyte migration (Basu and Minta, 1976). In the present study, the period for leucocyte sampling (up to 6 hr) was too short for any blood vessels to grow into the cornea (Basu, Ohashi and Hay, 1978). Therefore, any leucocytes t h a t were found in the corneal wounds during this period [Fig. l(b)] must have migrated from the limbal and conjunctival blood vessels either via the tear film (Norn, 1968) (extracorneal route) or through the avascular cornea surrounding the wound (intracorneal route) or both (Robb and Kuwabara, 1962; Basu and Minta, 1976). Initially (as seen in the 2 hr samples) the tear was the main route. Late PMNs came via both routes. Robb and K u w a b a r a (1962) showed that no leucocyte would invade the cornea by the intracorneal route before 5 or 6 hr following a corneal injury. In our experiment, however, we found that it was possible for the intracorneal invasion to occur before four hours after the injury (Fig. 2). Histological specimens taken at 2 hr did not show any leucocytes in the cornea intervening the wound site and the limbus. Therefore, all the PMNs observed at this time in the lesion (central and peripheral) must have come via the extracorneal route. As shown in Table I, irrespective of the group, the number of PMNs (as ascertained by the impression technique in the 2 hr samples) in the peripheral lesion was greater than that in the central lesion. The reason for this difference is not clear to us. I f it were true t h a t the PMNs after migrating from the limbal blood vessels formed a uniform cellular suspension in the tear which in turn bathed the entire corneal surface uniformly, why would then the number of PMNs in the wounds in the two locations vary ? Was it then possible t h a t the concentration of PMNs in the tear bathing the peripheral cornea was greater than that bathing the central cornea, or that some PMNs had crept along the epithelial surface (instead of going into the tears) and thus invaded in greater numbers the peripheral lesion which was closer to the site of their emigration from the limbal blood vessels ? However, after 2 hr following the injury (as seen in 4 and 6 hr histology specimens) when the PMNs started migrating through the intracorneal route, the lesions close to the limbus (peripheral wound) received, as expected, more PMNs than t h a t which was at a distance (the central wound). In the 2 hr specimens (when the route of PMN invasion into the corneal wound was exclusively extracorneal) in all of the 216 corneal wounds (both peripheral and central) t h a t we studied, the PMNs reached a distance up to about 0"5 m m laterally from both edges of the wound and vertically up to a depth of about 0-1 m m from the tip of the wound. The present study thus confirms the in vivo study of Robb and K u w a b a r a (1962) and our previous in vitro observations (Basu and Minta, 1976) that when leucocytes invade from the epithelial side through breaks in the surface layer, the PMNs can move laterally as well as deeper into the stroma. This phenomenon would suggest t h a t some substance(s) might have been liberated and diffused into the tissues surrounding the corneal wound, thus attracting the PMNs suspended in the tears beyond the furrow of the trephine incision [Fig. l(c)]. I f the presence of the PMNs in the lesion had involved only a passive process, the cells should have been confined only to the cavity of the wound and not beyond overcoming the resistance of the stroma. Thatcher, Darouger and Jones (1977) used polystyrene cover slips to impress inflamed conjunctival surfaces for collecting leucocytes. F r o m a study of different kinds of plastic materials, we found that Thermanox cover slips were among the best

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materials for collecting leucocyte samples from the conjunetival surface (Hershenfeld et al., 1981). The present study showed that this material was also quite suitable for studying the rate of mobilization of leucocytes into the corneal wounds. Leucocytes adherent to the cover slips could be readily stained, identified and counted following staining by the Diff-Quik technique (Herschenfeld et al., 1981). In view of the reports that alcohol reduces the stickiness of PMNs, it was interesting to note that many PMNs from the intoxicated rabbits actually remained adherent to the cover slips before their fixation. I t is, therefore, probable that alcohol affected some PMNs more than others. Obviously, due to the effect of alcohol on their adherance, many leucocytes could not come out from the limbal blood vessels (as leucocytes must adhere to the vascular endothelium before they can emigrate to the extravascular space), but those that came out must have had an optimum adhesiveness for their attachment to the cover slips. In the present experiment, we injected ethanol in doses which produced an alcohol concentration of about 600 mg/100 ml of blood. This dose did not kill the animals (n = 6) for at least up to 6 hr. In a preliminary experiment using a different group of rabbits, we injected alcohol in identical doses without doing any further experiments on them. All of these rabbits recovered in about 8 hr without any apparent ill effects. The doses we used (60 mg/100 ml of blood) would be potentially lethal to man as the lethal blood concentration in man has been found to range from 350 to 600 mg per 10 ml of blood (Mr D. M. Lucas, Director, Forensic Science Laboratories, Province of Ontario, pers. comm.). Our experiment thus shows that rabbits can tolerate ethanol better than man can. It has been demonstrated that in man when the blood alcohol level is as low as 150 mg/100 ml, there is an impairment of leucocyte mobilization into the skin wounds (Brayton et al., 1970). The lowest level of Mcohol that would produce a defect in the PMN mobilization into the corneal wound is not known at the present. We plan to investigate this and the relationship between PMN infiltration and corneal vascuiarization in the future. ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and the Lions' International District A-16 Ophthalmology Research Foundation. REFERENCES Basu, P. K. and Minta, J. O. (1976). Chemotactic migration of leucocytes through corneal layers. An in vitro study. Can. J. Ophthalmol. 11,235-40. Basu, P. K., Ohashi, K. and Hay, J. B. (1978). Blood flow in rabbit eyes after keratectomy and corneal transplantation. Can. J. Ophthalmol. 13, 89-92. Brayton, R. G., Stokes, P. E., Schwartz, M. S. and Louria, D. B. (1970). Effects of alcohol and various diseases on leucocyte mobilization, phagocytosis and intracellular bacterial killing. N. Engl. J. Med. 282, 123-8. Buckley, 1~. M., Ventura, E. S. and MacGregor, R. R. (1978). Propmnolol antagonizes the anti-inflammatory effect of alcohol and improves the survival of infected intoxicated animals. J. Clin. Invest. 62, 554-9. Gluckman, S. J. and MacGregor, R. R. (1978). The effect of acute alcohol intoxication on granulocyte mobilization and kinetics. Blood 52, 551-9. Hershenfeld, S., Kazdan, J., Mancer, K., Feugas, P., Basu, P.K. and Avaria, M. (1981). Impression cytology in conjunctivitis. Can. J. Ophthalmol. 16, 76-8. Issekutz, A. C. and Movat, H. Z. (1980). The in vivo quantitation and kinetics of rabbit neutrophil leukocyte accumulation in the skin in response to chemotactic agents and Escherichia coll. Lab. Invest. 42, 310-7.

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3ohnson, E. D. Jr (1975). Impaired defence mechanisms associated with acute alcoholism. Ann. N.Y. Acad. Sci. 252, 343-7. Louria, D. B. (1963). Susceptibility to infection during experimental alcohol intoxication. Trans. Assoc. Am. Physicians 76, 102-12. MacGregor, R. R., Gluckman, S. J. and Senior, J. R. (1978). Granulocycte function and levels of immunoglobulins and complement in patients admitted for withdrawal from alcohol. J. Infect. Dis. 138, 747-53. MacGregor, R., Spangnuolo, P. and Lentnek, A. (1974). Inhibition of granulocyte adherence by ethanol, prednisone and aspirin. Measured with an assay system. N. Engl. J. Med. 291,642-5. Maine, G., She~, S.M. and Leventhal, M. (1969). Endothelial contraction induced by histamine-type mediators. J. Cell. Biol. 42, 647-72. Marchesi, V.T. and Florey, H.W. (1960). Electron micrographic observations on the emigration of leucocytes. Q. J. Exp. Physiol. 45, 343-8. Moses, J. M., Gesehickter, E. H. and Ebert, g. H. (1968). Pathogenesis of inflammation. The relationship of ethanol permeability to leukocyte mobilization in delayed inflammation. Br. J. Exp. Pathol. 49, 385-94. Norn, M. S. {1968). Cells and vacuoles in mucous thread of inferior conjunctival fornix. Acta Ophthalmol. 45, 1125-34. Phelps, P. and Stainlaw, D. (1969). Polymorphonuclear leucocyte mobility in vitro. I. Effect of pH, temperature, ethyl alcohol and caffeine using a modified Boyden chamber technique. Arthritis Rheum. 12, 181-8. Pickrell, K. L. (1938). The effect of alcoholic intoxication and ether anesthesia on resistance to pneumococcal infection. Bull. Johns Hopkins Hosp. 63, 238-60. l~obb, R.M. and Kuwabara, T. (1962). Corneal wound healing. I. The movement of polymorphonuclear leukocytes into corneal wounds. Arch. Ophthalmol. bS, 636-42. Snyderman, R. and Mergenhagen, S.E. (1972). Characterization of polymorphonuclear leukocyte chemotactie activity in serums activated by various inflammatory agents. In Biological Activities of Complement (Ed. Ingram, D.G.). Pp. 117-32. Karger, Basel. Spagnuolo, P. J. and MacGregor, g. R. (1975). Acute ethanol effect on chemotaxis and other components of host defenee. J. Lab. Clin. Mad. 86, 24-31. Thatcher, R. W., Darouger, S. and Jones, B. R. (1977). Conjunctival impression cytology. Arch. Ophthalmol. 95, 678-81. Tisman, G. and Hebert, V. (1973). In vitro myelosuppression and immunosuppression by ethanol. J. Clin. Invest. 52, 1410-4.