Arch. 0to-Rhino-Laryng. 211, 177--184 (1975) �9 by Springer-Verlag 1975

Alkaline Phosphatase Activity in the Inner Ear Blood Vessels of Some Mammals

F u m i h i s a Hi ra ide , M. D., and Y a s u y a Nomura , M. D.

Department of Otolaryngology University of Tokyo, Tokyo, Japan

Received June 14, 1975

Summary. The distribution pattern of alkaline phosphatase in small blood vessels was studied histochemically in the stria vascularis and spiral ligament of various mammals. No or extremely weak activity of the enzyme was demonstrated in the walls of the blood vessels in bat and rabbit, while moderate to intense activity was found in guinea pig, cat, squirrel mokey and man. There was a species difference in the phosphate metabolism in the walls of the inner ear blood vessels.

The presence of a lkal ine phospha t a se a c t i v i t y in the vascu la r walls f var iouso organs was d iscovered i n d e p e n d e n t l y b y Gomori (1939) and T a k a m a t s u (1939). They found the enzyme a c t i v i t y local ized in the endo the l ium of capil laries, the endo the l ium of some smal l b lood vessels and a dve n t i t i a of med ium sized ar ter ies .

Subsequent h is toehemieal s tudies have been genera l ly in agreement wi th thei r obse rva t ions on the no rma l and a b n o r m a l an ima l t issues. I n these studies, how- ever, a v a r i a b i l i t y of enzyme a c t i v i t y of b lood vessels was no ted in var ious organs of the same an imal or in the same organ in different species of an imal .

The species difference in the i n t ens i t y of a lka l ine phospha t a se in the b lood vessels has been observed in some m a m m a l i a n cochleas b y N o m u r a and Hi ra ide (1968).

The p resen t s t u d y in tends to descr ibe the a lkal ine phospha t a se d i s t r ibu t ion p a t t e r n in the inner ear b lood vessels of ba t , r abb i t , guinea pig, cat , squirrel m o n k e y and m a n and to discuss b r i e f ly the possible role of this enzyme in the b lood vessels of the inner ear.

These inves t iga t ions would serve as basel ine d a t a in order to s t u d y the changes in this enzyme sys tem af ter different expe r imen ta l ma n ipu l a t i ons on the inner ear vascu la r sys tem of these an ima l species.

Material and Methods Three bats, 5 rabbits, 10 guinea pigs, 3 cats and 4 squirrel monkeys were used for the

present investigation. After decapitating these experimental animals under anesthesia by either intraperitoneal administration of nembutal or by an overdosis of ether, temporal bones were immediately removed from these animals and fixed in 4~ neutral formol calcium for 14 hrs at 4 ~ C. Human temporal bones were obtained at autopsies from patients who died of various diseases. For frozen section specimens the tissues were decalcified in a 5--10~ buffer solution of EDTA for 4 to 14 days at 4~ (Freiman, 1954). When decalcification was completed, the tissues were washed thoroughly for 10 rain in cold physiological saline solution. The decalcified temporal bones were frozen, mounted and cut serially at 15--20 ~z with a rotary microtome in a eryostat (--20~ Frozen sections were put on clean coverglasses, thawed slowly and dried at room temperature for 1/2--1 hr.

178 F. Hiraide and Y. Nomura

For the purpose of reactivating the enzyme (Freiman, 1954), the sections were placed in a i ~ solution of magnesium chloride from 2 to 4 hrs. Alkaline phosphatase was demonstrat- ed simultaneously by the azo-coupling method using naphthol AS-TR phosphate as a sub- strate and fast red violet LB salt as coupling agent (Burstone, 1958). Incubation periods varied from 4 to 12 hrs at 4 ~ C. Longer incubation periods did not change the results. Sections were counterstained with methyl green.

:For surface preparations the soft tissues were removed from the cochlea under dissection microscope. The tissues were incubated from 1 to 2 hrs in the same media as used for sectioned specimens. The specimens of surface preparations and sections were mounted in glycerine jelly or synthetic medium for light microscopic examination.

Findings Bat. No his tochemical reac t ion products appeared in the walls of any blood

vessels of the inner car of this an imal (Fig. l).

Rabbit. Most of the blood vessels in the inner ear of this an imal did no t show

any enzyme act iv i ty . However , considerable ac t iv i ty was demons t ra t ed in the

capil laries of spiral prominence.

Fig. 1. No alkaline phosphatase activity is seen in any vascular walls of the inner ear of bat. Considerable amount of azo dye products are noted in the cells of Reissner's membrane (RM) and the basal part of stria vascularis (SV). LSL lower spiral ligament, PC pigmented cells,

USL upper spiral ligament. Surface preparation

Alkaline Phosphatase Activity 179

Fig.2. Most blood vessels of the inner ear of rabbit do not show any enzyme activity. Con- siderable enzyme activity is occasionalty found in the capillaries of spiral prominence (NP), stria vascularis (SV) and arterioles of upper spiral ligament (USL). Some strial cells also

contain this enzyme. LSL lower spiral ligament. Surface preparation

Strong enzyme activi ty occurred occasionally in the par t of strial capillaries and arterioles of upper spiral ligament. The reaction products were visible frag- mental ly in their vascular walls (Fig. 2).

Guinea pig. An intense act ivi ty of alkaline phosphatase was seen in the walls of radiating arterioles and capillaries of the upper spiral ligament. All vessels in the lower spiral l igament (venules) revealed weak or negligible enzyme activity. The capillary blood vessels in the spiral prominence and stria vascularis showed varying degrees of enzyme activity. Some capillaries reacted strongly to alkaline phosphatase, while some did not, probably indicating that they are distal ends (Fig.3). The vas spirale showed the enzyme act ivi ty in its walls. The activity, however, was not uniform throughout the vessel. Moderate to weak enzyme activi ty occurred segmentally in the vessel under the tunnel. The small blood vessels in the other par t of inner ear demonstrated varying degrees of enzyme activity. I t was from strong to weak in segment.

Cat. Findings observed in the cat were almost similar to those seen in the guinea pig. However, some of the radiating arteriole showed very weak activi ty of the enzyme. Strong enzyme activi ty was found in a rolled network of capillaries in the spiral prominence (Fig.4A, B). The vas spirale was not present in the cochlea studied.

Monkey and Man. The histochemical findings in the monkey and human cochleas are essentially similar and will be described together.

Capillaries in the spiral l igament and stria vascularis showed strong activi ty of alkaline phosphatase. In contrast to guinea pig and cat cochlea, in which no

180 F. Hiraide and Y. Nomura

Fig. 3. An intense activity of alkaline phosphatase is noted in the walls of radiating arterioles and eapillaries of the upper spiral ligament (UNL) of the inner ear of guinea pig. All vessels in the lower spiral ligament (LSL) show weak or negligible enzyme activity. SP: spiral pro-

minenee, SV: stria vaseularis. Surface preparation

Fig. 4 A and B. Strong enzyme activity is found in a rolled network of capillaries in the spiral prominence (SP) of the inner ear of cat. Some of the radiating arterioles show weak activity of the enzyme. LSL: lower spiral ligament, S V: stria vascularis, USL: upper spiral ligament.

A: Surface preparation specimen. ~B: Sectioned specimen

enzyme ac t iv i ty was found in the venules of the lower spiral l igament , monkey and h u m a n eoehleas showed moderate to strong ac t iv i ty of the enzyme in the venules of the same area (Fig. 5). The capil lary below t u n n e l spaee showed ra ther

Fig~B.

Fig. 5. All the blood vessel~ in the lateral wall of human cochlea show strong alkaline phos- phatase activity. LSL: lower spiral ligament, SP: spiral prominence, SV: stria vaseularis,

USL: upper spiral ligament. Surfaee preparation

182 F. Hiraide and Y. Nomura

Table 1. Alkaline phosphatase activity in the inner ear blood vessels of various mammals. The intensities are expressed with the signs + + , +, • and --, which should be read as

intense, moderate, weak and no activity respectively

t~adiating Strial Spiral Collecting Spiral arterioles capillaries prominence venules vessel

v e s s e l s

:Bat - - - - - -

Rabbit -- -- + ~ • (occasionally (oeeasionMly +) +)

Guinea pig + + + -+ ~ • + + N + Cat + + N ~ : + + ~ • + + Squirrel monkey + + + + + + Man + + + + N + + +

• + + N ~

+ + + + + + N + + + N +

uniform activity of the enzyme throughout the segmental loop. Capillaries in the other part of inner ears were also clearly visualized.

Control specimens from each animal showed no histoehemieal reaction. The histoehemieM data obtained are summarized in Table 1.

Discussion

The present paper demonstrates the wide difference in the activity of alkaline phosphatase in small blood vessels of inner ears of different species using the identical method under standard condition.

No or little enzyme activity was detectable in the walls of inner ear blood vessels of the bat and rabbit. On the other hand, the small blood vessels of guinea pig, eat, squirrel monkey and man contained a remarkable amount of alkaline phosphatase in their walls.

The distribution pattern of this enzyme in the inner ear blood vessels was almost identical with that of blood vessels in the central nervous system. Accord- ing to Shimizu (1950), no enzyme activity was present in the blood vessels of the rabbit with the exception of only an occasional staining, while he found strong enzyme activity in the walls of brain blood vessels of guinea pig and eat.

Monoeha (1970) also reported high activity of this enzyme in the brain blood vessels of squirrel monkey. He is of the opinion that alkaline phosphatase might control the passage of various substances between the blood and brain tissue. I t is probable that alkaline phosphatase plays an important role in the transport of various chemicals and nutrients through the membranes of blood vessels to surrounding tissue cells. Samorajski and MeCloud (1961) indicated that the alkaline phosphatase plays an important role in the blood brain barrier mechanism.

There have been several reports of histoehemieal studies concerning alkaline phosphatase in the inner ear of various kinds of animals (Plotz and Perlman, 1955; Vinnikov and Titova, 1957; Raueh, 1964; Sehgtzle and Westernhagen, 1967; Nomura and Hiraide, 1969; Kirikae et al., 1969, and Hiraide, 1970). Plotz and Perlman (1955) first described the presence of this enzyme in the inner ear

Alkaline Phosphutase Activity 183

of bat. They did not find any detectable enzyme act ivi ty in the inner ear blood vessels of this animal. The present investigation on the localization of alkaline phosphatase act ivi ty in the inner ear of bat agrees with the results of their ob- servations. The positive reaction to alkaline phosphatase was seen in the basal par t of the stria vascularis adjacent to the spiral ligament. In the present study, however, considerable act ivi ty was also detectable in the cells of Reissner's membrane, plexus cochlcaris and on or near the spiral ganglion cells.

Later Vinnikov and Titova (1957) reported marked activi ty of this enzyme in the cells of the organ of Corti, the stria vascularis and other cells lining the endo- and peri lymphatic spaces of rabbit , guinea pig and cat. They neither described any detectable enzyme activi ty in the walls of inner ear blood vessels of these animals except the stria] vessels, nor mentioned the difference in the activity of this enzyme among animal species. However, the present s tudy has demonstrated remarkable species differences in enzyme distribution in the inner ear blood vessels and other inner ear tissues of rabbit , guinea pig and cat. Furthermore, their findings contradict greatly with those obtained from the present investigation. This difference may be at t r ibuted to the technique rather than enzyme distri- bution. The alkaline phosphatase distribution pat tern of the inner ear of guinea pig was generally identical with tha t previously reported by Sch~tzle and Western- hagen (1967). However, they did not describe the difference in the enzyme act ivi ty between arterial and venous small blood vessels. I t is evident in the present preparations, as stated earlier by Nomura and Hiraide (1968), tha t arterioles and proximal capillaries in the inner ear of guinea pig show high con- centrations of alkaline phosphatase, whereas venous vessels are negative, in- dicating the physiologic difference between arterial and venous blood vessels of this animal. In the inner ear of cat and squirrel monkey, the high act ivi ty of alkaline phosphatase is generally localized in the blood vessels and the connective tissues surrounding the nerve t runk (plexus cochlearis). The alkaline phosphatase in the blood vessels of these animals is particularly prominent in areas of spiral and vestibular ganglion cells, where blood vessels are more numerous.

The distribution pat tern of this enzyme in the inner ear of monkey shows fairly close similarity with tha t observed previously in human ears by Nomura and Hiraide (1968). In the inner ears of monkey and man, especially, the apparent selective localization of alkaline phosphatasc in the walls of small blood vessels stimulates speculation on the use of this enzyme for studying the course and distribution pat tern of capillaries.

This method has been employed to s tudy the course and distribution of the human inner ear blood vessels with excellent result (Kirikae et al., 1969).

We thus conclude tha t depending upon each species there is a difference in the phosphate metabolism in the walls of the inner ear blood vessels.

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Fumihisa I-Iiraide, M. D. Department of Otolaryngology Faculty of Medicine University of Tokyo Hongo, Bunkyo-ku, Tokyo Japan