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Effects of ischemia on the function and structure of the cat carotid body
Brain Research, 270 (1983) 63-76
63
Elsevier
Effects of Ischemia on the Function and Structure of the Cat Carotid Body L. MONTI-BLOCH*, L. J. STENSAAS and C. EYZAGUIRRE**
Department of Physiology, University of Utah, School of Medicine, Research Park, Salt Lake City, UT84108 (U.S.A.) (Accepted November 30th, 1982)
Key words: ischemia - carotid body - chemosensory discharge
Interrupting the blood supply to the carotid body by ligating arteries of its vascular peduncle altered the chemoreceptive properties of the carotid nerve and produced structural changes in parenchymal (glomus and sustentacular) cells. The onset ofischemia was marked by an increase in the discharge of both A (myelinated) and C (unmyelinated) sensory fibers followed by depression and finally by receptor silence. The discharge of A-fibers disappeared after 30-50 min and that of C-fibers after 60-90 min. During ischemia of 15-60 min duration the threshold to pharmacological (NaCN, ACh) and 'natural' (hypoxic) stimuli progressively increased and was accompanied by reversible changes in the structure of parenchymal cells and nerve endings, lschemia for 2 h or longer produced irreversible functional damage and disappearance of glomus and sustentacular cells from the carotid body. Following ischemic injury, nerve fibers regenerated and all responded to mechanical stimuli but only a few were stimulated by natural or pharmacological agents. Thus, parenchymal cells of the carotid body appear to be most important in transduction by allowing sensory fibers to respond to chemical stimuli. INTRODUCTION
The carotid body is a complex sensory receptor formed by myelinated (A) and unmyelinated (C) sensory fibers j3 which innervate the glomus cells in the organ. The glomus cell-nerve ending junction is enveloped by sustentacular cell processes. For a number of years it has been assumed that integrity of the whole receptor complex (cells and nerve fibers) is essential in the transduction process This view has been supported by several investigators who have indicated that sensory fibers of the carotid nerve are incapable of normal chemoreception without the presence of carotid body cells7,33,38.39,43.Others have suggested the opposite, namely, that the parenchymal cells are not essential components of this process3,24. 27.
In view of these discrepancies we sought to clarify the role of carotid body cells in transduction by using acute and chronic ischemia to destroy them. These experiments revealed that total short-term ischemia (60 min or less) induced
functional and morphological alterations that recovered after restoring circulation or placing the preparations in vitro. However, arterial occlusion for 2 h or longer led to irreversible functional and morphological damage to the chemoreceptors whose response to natural and pharmacological stimuli disappeared after ischemia. Our results thus agree with those of others who eliminated the carotid body by cryodestruction39 or who removed it surgically in the cat38 or human2~. It appears, therefore, that carotid body parenchymal cells are important in chemotransduction. They seem to condition the afferent fibers to respond to chemical stimulation. Some preliminary results have been published elsewhere29. MATERIALS AND METHODS
Surgical procedures for the production of carotid body ischemia Experiments were performed in 43 adult cats anesthetized with sodium pentobarbitone (40
* Present address: Departamento de Fisiologia, Facultad de Medicina, Universidad de la Repfiblica, Montevideo, Uruguay, ** To whom correspondence should be addressed. 0006-8993/83/$03.00 ,,~ 1983 Elsevier Science Publishers B.V.
64 m g / k g i.p.) and heparinized (500 U/kg, iv.). The right carotid body was identified and the vascular peduncle which provides the arterial supply to the organ was exposed. A ligature consisting of 6-0 silk thread was placed around the vascular peduncle; ligatures were also situated on the occipital and the ascending pharyngeal arteries close to the carotid body (Fig. 1, arrows). The veins draining the organ were left intact. Four groups of experiments were performed: (1) in 22 cats, acute ischemia (15 min to 6 h) was produced and the ligatures were left in place; (2) in 9 animals, acute ischemia was followed by short-term recovery (30 min to 4 h), by removal
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cca Fig. 1. Schematic drawing of carotid body region and ligated blood vessels (arrows): cca, common carotid artery; eca, external carotid artery; ica, internal carotid artery; oca, occipital artery; aph, ascending pharyngeal artery; cb, carotid body; sn, sinus (carotid) nerve; scg, superior cervical sympathetic ganglion; cv, carotid body vein.
of the ligatures or placing the carotid body in vitro and superfusing it with oxygenated saline; (3) in 6 preparations, acute ischemia was followed by prolonged recovery, by removing the ligatures for 18-48 h; (4) in 6 cats, chronic ischemia (12 h to 10 months) was produced by leaving the ligatures in place. Operations were performed under aseptic conditions when the intended survival period was 18 h or longer.
Electrophysiology In situ recordings were made from both normal and ischemic carotid body preparations. The ganglio-glomerular nerves were cut and baroreceptor fibers were crushed around the carotid sinuses to eliminate mechanoreceptor activity ~2. The carotid nerve was surgically isolated, sectioned close to the glossopharyngeal nerve, and protected from drying by warm mineral oil. Action potentials were recorded from the nerve with platinum-iridium electrodes connected to a conventional AC recording system. Nerve discharges were stored on FM tape (frequency response, DC to 1250 Hz) for analysis and photography. Frequency changes of sensory units, selected with a window discriminator, were measured with a counter and printer system and fed to a digital-analog converter for display on a chart recorder or oscilloscope. Conduction velocity of afferent fibers in small filaments containing one or few active units was estimated by utilizing a double set of recording electrodes (5 m m separation) to measure differences in arrival time of the same action potentials ~3. This method gives only an approximate estimate of conduction velocity in different fiber groups, because of the short length of nerve between the pairs of recording electrodes 34. Chemosensory responses to systemic or 'topical' application of natural and pharmacological stimuli or inhibitors were studied. Hypoxia was produced by inhalation of 100% N2 or by gently blowing this gas over the carotid body for 1-2 min, using a 23-gauge needle placed close to the organ and connected to the gas reservoir. For hyperoxia the same procedures were used for 100% 02. Nicotine bitartrate (K and K), NaCN (Merck) and dopamine HC1-DA (Sigma) dis-
65 solved in modified Tyrode's solution were either injected into the femoral vein or applied directly (using a microdispenser) to the carotid body in volumes of 5 /~1. Acetylcholine chloride-ACh (Sigma) was always applied directly. The topically applied drugs were removed by repeated rinsing of the preparation with 0.25 ml of warm saline. In vitro recordings were obtained from 7 ischemic preparations. The carotid bodies and their own nerves were dissected out and placed in a chamber through which flowed modified Tyrode's solution (pH 7.43) at 36 °C. Gases and drugs were delivered by using methods previously reported s,~l.~,_2s. Electron microscopy Tissue was prepared for ultrastructural analysis with a fixative containing 0.1 M phosphate buffer (pH 7.4), 1% paraformaldehyde and 1% glutaraldehyde. Carotid bodies with ligatures situated on the vascular peduncle were removed and fixed by immersion for 8 12 h. Preparations with an intact vascular supply were perfused at a pressure of 100 torr for 10 min, the carotid bodies removed and placed in cold fixative for 8- 12 h. All tissue was rinsed in 2.5% NaC1, postfixed in 2% osmic acid in 0.1 M phosphate buffer for 2 h, dehydrated in a graded series of alcohol and embedded in Epon. Semithin sections were cut through the mid-portion of the carotid body. The blocks were retrimmed; ultrathin sections were cut, mounted on one-hole (1 mm x 2 mm) grids on Formvar film and stained with uranyl acetate and lead citrate. Reactive changes in intralobular and extralobular constituents of the carotid body were determined electron microscopically by systematic examination of single ultrathin sections through the mid-portion of the carotid body. The evaluation of each constituent was facilitated by the use of one-hole grids which afforded an unobstructed view of the entire section. Twenty-five glomus cells in which the nucleus was visible were selected for a detailed examination of reactive changes. The axon terminals, sustentacular cells and unmyelinated intralobular axons associated with these cells were subsequently inspected. Finally, myelinated and unmyelinated
axons between the lobules containing the glomus cells were systematically examined for reactive changes. Information was then tabulated: (0) indicated absence or necrosis of a given element, ( - ) few reactive constituents, ( - - ) many reactive constituents, ( + ) normal incidence of a given element, and ( + + ) supra-normal incidence. RESULTS
The effectiveness of the ligatures in producing ischemia was evaluated morphologically. Of 43 carotid bodies examined with the electron microscope, 22 (51%) showed unequivocal signs of total ischemia while the rest (49%) showed that ischemia was incomplete. The reasons for such variability are unknown since an effort was made to tie all visible arterial vessels entering the carotid body. On two occasions, both arterial and venous vessels were tied, resulting in faster and more profound changes than when only the arteries were ligated. This combined procedure was not employed routinely because of the possibility of damaging the carotid nerve. Physiological observations A cute ischemia Ligature of the carotid body vascular peduncle resulted in an increase in frequency of the chemosensory discharge, which reached a peak in about 5 min; this was followed by a progressive decrease, until complete silence was attained at nearly 90 min (Fig. 2). When sensory units with different spike amplitudes were analyzed through the window discriminator, responses with different temporal patterns were observed. Ischemia induced a significantly (P 0.01) larger increase in frequency for sensory units with larger spikes (A), than for those with small spikes (B). Also, the fibers with larger spikes (A) ceased firing after 3(L50 min of continued ischemia, whereas those with small spikes (B) did so 60-90 min after applying the ligature. Conduction velocity measurements showed that the low amplitude action potentials corresponded to sensory fibers conducting at 0.5-0.9
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Fig. 2. Frequency of spontaneous chemosensory discharges recorded from carotid nerve after tying carotid body vascular peduncle. Filled circles and triangles, mean frequency ( ± S.E.) during 60 s recordings. A and B, two different populations ofchemosensory fibers with large and small spikes, respectively. C, control discharge levels.
m/s, while the larger spikes occurred in sensory units conducting at 5-10 m/s. These conduction velocities are comprised within the ranges reported for A and C carotid nerve chemosensory fibers from normal carotid body preparations 13. When hypoxic stimulation (100% N2) was topically applied (Fig. 3) the discharge of the normal carotid nerve increased markedly (A). These fibers became almost silent after 60 min of ischemia (B), but another topical application of 100% N 2 w a s still capable of inducing some receptor stimulation (C). The responses to topical application of stimulants such as ACh 25-100 #g or NaCN 10/~g and the inhibitor DA 100/~g35m were depressed after 15-30 min ischemia and absent when the preparation was ischemic for 60 min or more The fact that DA failed to depress discharges when ap-
plied to 60 min ischemic preparations may have been due to the very low basal discharge (Fig. 2) with little room for further depression Fig. 4 illustrates the dose-dependent responses to ACh of normal preparations (open circles) and preparations subjected to ischemia. Relatively short ischemia (15 min) depressed all responses to ACh, whereas only a minimal response to the largest doses of ACh (100 ~g) was elicited after 30 min ofischemia; all responses to the drug were abolished after 60 min of ischemia. During ischemia there were variations in the response of different preparations to each type of stimulus. At times, the response to ACh was eliminated after 30 min ofischemia while that to 100% N 2 still occurred. In other instances the opposite was true.
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Fig. 3. A: carotid nerve discharges during topical 100%N 2 to normal carotid body. B: spontaneous chemosensoryactivity of same preparation after 1 h ischemia. C: 100%N 2 applied after I h ischemia.
Results just presented refer to preparations in which, morphologically, complete ischemia was established. Preparations in which ischemia was incomplete were still able to respond to inhaled N 2 and to systemic injections of N a C N , nicotine and DA for several hours after ligature of the vascular peduncle. An interesting feature occasionally observed was that a preparation partially ischemic for 45 60 min apparently became hyper-reactive to different stimuli. For instance,
100% N2 inhalation induced a peak/baseline discharge ratio of 1.1 (dF = +38.3 Hz) in the normal preparation with a mean baseline discharge of 156 Hz. Partial ischemia produced a great initial increase in chemosensory discharges, but after 60 min, the mean baseline frequency decreased to 28.1 Hz and during 100% N 2 inhalation the peak/baseline ratio increased to 13.1 (dF = +87.3 Hz). Similar results were obtained in the same preparation during topical applications of ACh 25/~g. Before ischemia the peak/baseline ratio was 1.5 (dF = 85.7 Hz), which increased to 2 2 (dF = 340 Hz) after 60 min of ischemia. The reasons for these differences are unknown and require further investigation Chronic ischemia Chronic ischemia was produced by ligation of the vascular peduncle for periods ranging from 12 h to 10 months. The carotid body appeared
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Fig. 5. Carotid nerve discharges from long-term ischemic carotid bodies. A, small action potentials from 2-day ischemic glomus; B, small and larger spikes in 7-day ischemic preparation: C, local tapping of 7-day ischemic glomus induces burst of action potentials: D, spontaneous discharges from I month ischemiccarotid body. swollen after 2 days, but could not be identified under the stereomicroscope (6-25 x ) after 7 days. A great reduction in spontaneous activity was observed after 2 and 7 days of complete ischemia, and consisted of very small action potentials and a few larger spikes (Fig. 5A and B). Topical application of 100% N2, ACh 100 ~g, nicotine 64 ~g, NaCN 10 /~g and DA 100 /xg usually did not change the spontaneous discharge frequency. Mechanical stimulation of the tissue attached to the carotid nerve in the 7-day preparation gave rise to short bursts of activity (Fig. 5C). Ischemia for 1 month resulted in a very marked reduction in spontaneous activity, consisting of both very small and somewhat larger spikes (Fig. 5D). The experiment illustrated in Fig. 5D was analyzed to compare nerve activity in the ischemic and normal carotid body. The carotid nerve of this 1-month ischemic preparation had a basal discharge of 1.4 _+0.3 Hz (mean + S.E.) as opposed to 157 + 2.6 Hz on the normal side. This indicates that the operated side had an activity which was 0.09% of normal. Stimulation by inhalation of 100% N, showed that the reacti-
vity of the few units remaining in the operated side was not significantly different from the normal preparation. In fact, the mean peak/baseline discharge ratio was 3.76 on the operated side vs 1.21 on the normal preparation (P 0.2). When 100% 02 was inhaled these ratios were 0.88 and 0.54 respectively (P ~ 0.001) indicating that 100% 02 was more effective on the normal preparation. Differences between the effects o f N 2 and 02 tests may have been due to the very different baseline discharge in both cases9. Ultrastructural control of this ischemic preparation revealed an absence of both glomus cells and sensory nerve terminals, but an abundance of unmyelinated axons. It is possible that during this long ischemic period regenerating axons may have innervated some of the numerous miniglomera located near the carotid body, which continued to respond to the usual chemosensory stimuli 26. Alternatively, a few regenerating chemosensory fibers may have been able to respond directly to the aforementioned stimuli. In fact, regenerating mammalian fibers in other systems show spontaneous activity which can be modified by substances such as adrenaline 5,6,36.
69 Chronic partial ischemia (1 day-10 months) usually induced a depressed response to different stimuli. For example, one partially ischemic carotid body kept for 10 months was tested with topically applied 100% N2, N a C N 10 /zg, ACh 100 ~g and DA 100/~g. The peak/baseline ratios obtained from the operated side were 52% for N2, 32% for NaCN, 14% for ACh and 40% for DA of the ratios recorded from the contralateral normal cartid body. Similar reductions were observed when dF values were compared. These observations indicate that a prolonged ischemic insult may produce permanent physiological damage to the glomus-sustentacular complex even though it may survive as a morphological entity. In all probability nerve fibers injured during this process had ample time to regenerate.
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Restoration offunction after acute ischemia Short-term recovery. Preparations totally ischemic for 15 min to 2 h either had the vascular ligatures removed or were placed in vitro to study possible recovery of chemoreceptor function. Results were similar in both experimental situations. After a 30-min ischemic period the responses to 100% N: or NaCN 5 /~g were markedly reduced and recovered only partially either in vitro or after restoring circulation. The depressed responses to ACh 100 ~g or DA 40 ~tg topically applied were not restored. Ischemia for 60 min resulted in a significantly reduced response to 100% N 2. Placing the preparation in vitro or restoring circulation for 1 h induced partial recovery only in some preparations (Fig. 6). As shown before, applications of
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70 NaCN 10/.% ACh 100 ~g and DA 100 #g were ineffective in changing the sensory discharge frequency after 60 min ischemia. These effects failed to recover in vitro or after removing the ligatures for 1 h. Long-term recovery. Spontaneous activity consisting only of small spikes was present in preparations allowed to recover for 2 days following 60 min ofischemia. These fibers had conduction velocities of 0.4-0.7 m/s corresponding to he slower fibers of the intact carotid nerve. The frequency of discharges from the whole nerve was well below the discharge level of the contralateral normal carotid nerve. Natural or pharmacological stimuli did not change the activity of these preparations. However, mechanical stimulation of the carotid bifurcation induced volleys of small action potentials in fibers that were not spontaneously active. Inspection of the region showed either no visible carotid body or one which was quite small. TABLE
A 1 h ischemic preparation allowed to recover for 30 days showed no identifiable carotid body within the carotid bifurcation. The nerve contained few spontaneously active fibers producing small action potentials and a few spikes of higher amplitude with conduction velocities of 0.5-1.5 m/s. Applications of 100% N2, ACh 100 /~g and NaCN 10/tg were without effect. However, mechanical stimulation of the area produced short volleys of action potentials. These results indicate that chemoreceptor function altered by an ischemic insult can be partially restored if ischemia lasted 60 min or less. More prolonged periods of ischemia (2 h or longer) induced permanent damage to the chemoreceptor complex.
Morphological observations Reactive and degenerative changes following ischemic periods up to 12 h involved a progres-
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71 sive and time-dependent sequence. Table I summarizes the morphological findings concerning reactive changes in the parenchymal components of lobules and in axons of the extra-lobular compartment (stroma). The sequence of ultrastructural changes involved: axon terminal detachment (Fig. 8), degeneration of glomus cells (Fig. 10), retrograde degeneration of extralobular axons (Fig. 11), disappearance of glomus and sustentacular cells, and regeneration of axons into the depopulated connective tissue of the carotid body (Fig. 12). Reactive ultrastructural changes developed rapidly following short periods of ischemia. They were evident in all lobular constituents, and intially involved alterations in their appositional relations. Cytological changes in glomus cells appeared before alterations were detected in sustentacular cells, Schwann cells or axons (Figs. 7 9). The fact that changes followingshort-term ischemia with no recovery were vir-
Fig. 7. Fifteen min ischemia, 1 h recovery. Pror~inent extracellular spaces (asterisks) occur between glomus (GC) and sustentacular cell processes (arrows). Spaces minimally affect axon terminals which remain apposed to glomus cells in 'edematous' lobule. 7375 x .
tually identical to those of ischemia followed by short periods of recovery (1-4 h) indicated that such cytological alterations occurred rapidly. Changes occurring up to 2 h of ischemia were proportional to the duration of the ischemic period. The most conspicuous were those affecting the cytoplasm of glomus cells, and those involving detachment of sustentacular processes; such changes were apparent after only 15 min of ischemia and 1 h recovery (Fig. 7). The spaces which developed at the sites of detachment gave the lobules an 'edematous' appearance. In general, glomus cells in such lobules displayed a reduced incidence of granular vesicles and a cytoplasm markedly lighter than normal. However, in lobules lacking intralobular spaces some cells had a cytoplasm darker than normal; such cells also had an increased incidence of granular vesicles and were abnormal in size and shape. Major alterations in synaptic relations occurred after 30 min ischemia and 1 h recovery. They involved a substantial reduction in the area of apposition between nerve terminals and glomus cells in certain lobules. As shown in Fig.
Fig. 8. Thirty rain ischemia, 1 h recovery. Early stage ofaxon terminal (AT) detachment involves intercalation ofsustentacular cell processes (arrows) between axon and glomus cell (GC). Note unusual profusion ofsustentacular cell processes within intralobular spaces (circled). 6450 x .
72 8, thin lamellar processes of sustentacular cells were interposed between glomus cells and axonal enlargements identified as calyciform terminals. The process of axon terminal detachment through the interposition of a single lamella of a sustentacular cell was common in the non-edematous lobules. However, there were a few instances of multiple lamellae of sustentacular cells separating axon terminals from glomus cells with dark cytoplasm. Also, edematous spaces separated a few axon terminals and glomus cells with light cytoplasm. Ischemia for 1 h appeared to be at or near threshold for inducing death of parenchymal cells since some necrotic glomus cells were present in 1-h preparations following recovery times of 24 or 48 h. These carotid bodies also showed a profound reduction in the number of terminals apposed to glomus cells (Fig. 9) and a reduced incidence of intralobular unmyelinated axons. However, there was no reduction in the number ofmyelinated or unmyelinated axons in the stroma and no clear signs ofaxonal degeneration, although the cytoplasm of all axons was darker than normal. Reduction in the number and extension of synapses in the absence of Wal-
Fig. 9. One h ischemia, no recovery. Extensive detachment of axon terminals (AT) occurs by intercalation of multiple reactive sustentacular cell processes (arrows) between axon and glomus cell (GC). 11,100 ×.
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Fig. 10. Two h ischemia, no recovery. Necrotic glomus cell (GC) undergoing lysis, phagocytosis (arrows) and lysosomal disintegration within sustentacular cells (white asterisks). Note unapposed nerve profiles resembling axonal termirials (AT) and intralobular unmyelinated axons (black asterisks). 6450 ×.
lerian degeneration appeared to result from terminal detachment followed by withdrawal of the axon from the lobule, possibly involving retraction into interlobular myelinated axons within the stroma. Carotid bodies subjected to 2 h of ischemia without a recovery period contained many necrotic glomus cells. The 2-h period was above threshold for inducing cell necrosis. Both 1- and 2-h ischemic preparations with 1 h of recovery displayed extensive reactive changes in the cellular constituents of all lobules. Intralobular axons having the cytological characteristics of terminals but lacking contact with glomus cells were common. All were surrouncaed by multiple lamellate processes arising from reactive sustentacular cells. Cell necrosis after 2 or more hours of ischemia involved lysis of light glomus cells and pyknosis of the dark cells. Fragmentation and disintegration of a glomus cell induced an
73 immediate phagocytic response in nearby sustentacular cells (Fig. 10). Preparations recovering for more than 1 h after 1 and 2 h ofischemia contained few axon terminals and few intralobular unmyelinated axons, but the incidence of extralobular myelinated axons was normal. Thus, the loss of intralobular axons through carotid body ischemia appears to be related to reactive changes leading to the disintegration and disappearance of glomus cells. Reactive changes in extralobular myelinated axons included shrinkage and the development of dark cytoplasm with irregular cisternae. Dense-core vesicles were somewhat more numerous in the axons of nerves which had been cut to permit recording of action potentials than in intact nerves. Parenchymal elements in various stages of disintegration and removal were present in 2-h ischemic preparations recovering for 12 h. Disappearance of the lobular constituents left connective tissue in which there were reactive nerves, granular leucocytes, fibroblasts, and reactive sinusoid capillaries. Loss of axoplasm was characteristic of interlo-
Fig. 11. Twelve h ischemia, no recovery. Interlobular unmyelinated (U) and myelinated axons (M) in association with necrotic glomus cell (GC). 6340 x .
Fig. 12. Seven day ischemia, no recovery. Axons (A) resembling regenerating axon sprouts occur in the carotid body in association with Schwann cells. 11,100x.
bular myelinated axons following 12 h of ischemia; however, many axons survived in spite of a persisting impediment to circulation (Fig. 11). Axons having the fewest reactive changes were those invested by an intact and well developed perineurial sheath. Regenerating unmyelinated axons were present in the carotid body of animals which survived for 7 and 16 days after 2 or more hours of ischemia. They appeared to originate from myelinated axons which had undergone few retrograde changes. The cytoplasmic characteristics of regenerating axons in the carotid body stroma were identical to those of the regenerating peripheral nerve (Fig. 12). However, perineurial components were either absent or lacking around such axons and there was little or no 'compartmentation '3~-32. In all cases, axons occurred in association with Schwann cells. Persistence of unmyelinated axons resembling axon sprouts together with numerous myelinated axons in animals surviving 30 days or more indicate both continuing axonal growth and remyelination in the absence of glomus cells. The unmyelinated axons were observed to have neither an external configuration characteristic of normal intralobular axons nor to contain the aggregates of vesicles typical of terminals in the normal carotid body.
74 DISCUSSION
Heymans and Bouckaert 2° and Alvarez-Buylla ~ altered the chemoreceptor properties of the carotid body by occluding the circulation within the organ using injections of Lycopodium spores or talcum powder. Alvarez-Buella ~reported that such occlusion induced an increased carotid nerve chemosensory discharge lasting 15 min, followed by disappearance of spontaneous chemosensory activity and elimination of chemoreceptor responses, while leaving barosensory activity intact. Nishi et al. 33 surgically occluded blood vessels to the carotid body and found similar changes. They observed recovery after restoring circulation to the preparation, provided that the ischemic period lasted no longer than 60 min. Our results are in essential agreement with all of the earlier studies. Whether the reduction in chemosensory function was due primarily to alterations in glomus cells or to changes in the sensory nerve terminals could not be determined from the ultrastructural changes following short-term ischemia (less than 60 min), since both were altered. Cytological changes of sensory nerve terminals were generally less conspicuous than those of glomus cells; but since the morphological integrity of both elements was compromised, it suggests that both may have been involved. The morphological changes correlated well with the physiological findings following both long-term ischemia (up to 30 days) and ischemia of 60 min or more with long-term recovery. In these cases glomus cells became necrotic and disappeared while the nerve regenerated. Our finding that the great majority of sensory fibers in such preparations lacked chemoreceptive properties contradicts the suggestion of others that glomus cells are not required for chemotransduction24,27. Present results are in accord with the findings ofVerna et al? 9 who destroyed parenchymal cells of the carotid body by freezing and allowed time for the nerve to regenerate: such preparations showed no chemosensitivity when cryodestruction was complete. Our conclusions also agree with the findings of Smith and Mills 38 who removed both carotid bodies
and showed no carotid nerve chemoreceptor activity in animals tested several months later. Similarly, studies in humans following bilateral removal of the carotid body to alleviate asthma showed severe impairment of the ventilatory hypoxic response 2j. Therefore, most of the available evidence indicates that glomus cells are necessary for chemotransduction and that regenerated or regenerating carotid nerve fibers cannot perform this function alone. This agrees with the experiments of Zapata et al. 43, who crushed the carotid nerve and found no chemoreception until the regenerating fibers became apposed to carotid body parenchymal cells. Our experiments further suggest that peripheral, preneural elements (glomus cells in this case) are capable of 'conditioning' the sensory nerve fibers to respond to particular sensory stimuli. Such changes in the functional properties of axons may involve transfer of a chemical signal from the glomus cell to the perikaryon of the sensory neuron via retrograde axonal transport. However, no detailed analysis of changes in centripetal transport of materials before and after removal of glomus cells has been performed. It is known that the carotid nerve can transport horseradish peroxidase from the periphery to the sensory (petrosal) ganglion and central processes2,4,23 as it can transport radiolabeled amino acids from this ganglion to the carotid b o d f 4-37.Thus, while there is no direct evidence that retrograde transport may influence petrosal sensory neurons and render its endings chemosensitive, differences between the electrical properties of chemo- and barosensory ganglion cells indicate this possibilitf 6. If a peripheral contribution is required to condition the behavior of sensory neurons, the situation may be similar to that of mammalian motoneurons some of whose properties are dependent on the muscle cells which they innervate 15. Despite lack of information as to the means by which carotid body parenchymal cells influence sensory neurons, it is known that ACh and catecholamines are contained in these cells and are released during stimulation ~°,~2-~8-19.More recently, it has been found that glomus cells also contain enkephalins 25,4° and that substance P is
75 present in carotid nerve fibers 22 and petrosal ganglion cells 17. These chemicals, plus others still not identified, may be involved in trophic interactions between glomus cells and sensory neurons and may induce the carotid nerve afferents to behave as chemoreceptor elements. This possibility is explored further in the following paper where the carotid body was grafted into a muscle and innervated with fibers from the muscle nerve3°.
ACKNOWLEDGEMENTS
We wish to express our appreciation to Dr. P. Zapata for reading this manuscript and providing valuable suggestions. We are also grateful to Messrs. P. Lenoir, J. Fisher and B. Evans for technical assistance. Mrs. Carolina Zapata helped in the preparation of this paper and illustrations. Work sponsored by Grants NS-05666 and NS-07938 from the U.S. Public Health Service.
REFERENCES 1 Alvarez-Buylla, R., Disociaci6n de las actividades qui-
2
3 4 5
6 7
8
9 10 11 12
13 14
miorreceptoras y barorreceptoras en gatos, Arch. Inst. Cardiol. Mex., 24 (1954) 26-37. Berger, A. J., The distribution of the cat's carotid sinus nerve afferent and efferent cells bodies using the horseradish peroxidase technique, Brain Research, 190 (1980) 309-320. Biscoe, T. J., Carotid Body: Structure and Function, Physiol. Rev., 51 (1971)427 495. DeGroat, W. C., Nadelhaft, I., Morgan, C. and Schauble, T., The central origin of efferent pathways in carotid sinus nerve of the cat, Science, 205 (1979) 1017 1018. De Santis, M. and Duckworth, J. W., Properties of primary afferent neurons from muscle which are spontaneously active after a lesion of their peripheral processes, Exp. Neurol., 75 (1982) 261- 274. Devor, M. and Jiinig, W., Activation of myelinated afferents ending in a neuroma by stimulation of the sympathetic supply in the rat, Neurosci. Lett., 24 ( 1981) 43-47. Eyzaguirre, C. and Fidone, S. J., Transduction mechanisms in the carotid body: glomus cells, putative neurotransmitters and nerve endings, Amer. J. Physiol., 239 (Cell Physiol. 8), (1980) C135 C152. Eyzaguirre, C. and Koyano, H., Effects of hypoxia, hypercapnia, and pH on the chemoreceptor activity of the carotid body in vitro, J. Physiol. (Lond.), 178 (1965) 385409. Eyzaguirre, C. and Koyano, H., Effects of electrical stimulation on the frequency of chemoreceptor discharges, J. Physiol. (Lond.), 178 (1965) 438-462. Eyzaguirre, C., Koyan6, H. and Taylor, J. R., Presence of acetylcholine and transmitter release from carotid body chemoreceptors, J. Physiol. (Lond.), 178 (1965) 463 476. Eyzaguirre, C. and Lewin, J., Effect of different oxygen tensions on the carotid body in vitro. J. Physiol. (Lond.), 159 (1961) 251-267. Eyzaguirre, C. and Zapata, P., The release of acetylcholine from carotid body tissues. Further study on the effects of acetylcholine and cholinergic blocking agents on the chemosensory discharge, J. Physiol. (Lond.), 195 (1968) 589-607. Fidone, S. J. and Sato, A., A study of chemoreceptor and baroreceptor A and C-fibres in the cat carotid nerve, J. Physiol. (Lond.), 205 (1979) 527-548. Fidone, S, J., Zapata, P. and Stensaas, L. J., Axonal
15
16
17
18 19
20
21
22 23
24 25
transport of labeled material into sensory nerve endings of cat carotid body, Brain Research, 124 (1977) %28. Gallego, R., Kuno, M., Nufiez, R. and Snider, S. W., Enhancement of synaptic function in cat motoneurons during peripheral sensory regeneration, J. Physiol. (Lond.), 306 (1980) 205-218. Gallego, R. and Belmonte, C., Electrical properties ofpetrosal ganglion chemoreceptor cells. In: C. Belmonte, H. Acker, D. Pallot and S. Fidone (Eds.), Arterial Chemoreceptors, Proc. VI Int. Meeting, Leicester Univ. Press, 1981, pp. 39~402. Gillis, R. A., Helke, C. J., Hamilton, B. L., Norman, W. P. and Jacobowitz, D. M., Evidence that substance P is a neurotransmitter of baro- and chemoreceptor afferents in nucleus tractus solitarius, Brain Research, 181 (1980) 476-481. Gonz~ilez, C. and Fidone, S., Increased release of3H-do pamine during low 02 stimulation of rabbit carotid body in vitro, Neurosci. Lett., 6 (1977) 95-99. Hanbauer, I. and Hellstr6m, S., The regulation of dopamine and noradrenaline in the rat carotid body and its modification by denervation and by hypoxia, J. Physiol. (Lond.), 282 (1978) 21 34. Heymans, C. and Bouckaert, J. J., Au sujet de la technique de la preparation du sinus carotidien circulatoiremerit isol6 mais nerveusement intact, C. R. Soc. Biol., Paris, 113 (1933)912-913. Honda, Y., Watanabe, S., Hashizume, I., Satomura, Y,, Hata, N., Sakakibara, Y. and Severinghaus, J. W., Hypoxic chemosensitivity in asthmatic patients two decades after carotid body resection, J. appl. Phvsiol., 46 (1979) 632-638. Jacobowitz, D. M. and Helke, C. J., Localization of substance P immunoreactive nerves in the carotid body, Brain Res. Bull., 5 (1980) 195-197. Kalia, M. and Davies, R. O., A neuroanatomical search for glossopharyngeal efferents to the carotid body using the retrograde transport of horseradish peroxidase, Brain Research, 149 (1978) 477-481. Kienecker, E. W., Knoche, H. and Bingmann, D., Functional properties of regenerating sinus nerve fibers in the rabbit, Neuroscience, 3 (1978) 977-988. Lundberg, J., H6kfelt, T., Fahrenkrug, J., Nilsson, G. and Terenius, L., Peptides in the cat carotid body (glomus caroticum): VIP-, enkephalin-, and substance P-like immunoreactivity, A cta physiol, scand., 107 (1979) 279 281.
76 26 Matsuura, S., Chemoreceptor properties ofglomus tissue found in the carotid region of the cat, J. Physiol. (Lond.), 235 (1973) 57-73. 27 Mitchell, R. A., Sinha, A. K. and McDonald, D. M., Chemoreceptive properties of regenerated endings of the carotid sinus nerve, Brain Research, 43 (1972) 681- 685. 28 Monti-Bloch, L. and Eyzaguirre, C., A comparative physiological and pharmacological study of cat and rabbit carotid body chemoreceptors, Brain Research, 193 (1980) 44%470. 29 Monti-Bloch, L., Stensaas, L. J. and Eyzaguirre, C., Effect of ischemia on carotid body structure and function. In: C. Belmonte, D. Pallot, H. Acker, and S. Fidone (Eds.), Arterial Chemoreceptors Proc. VI Int. Meeting, Leicester Univ. Press, Leicester, 1981, pp. 133-142. 30 Monti-Bloch, L., Stensaas, L. J. and Eyzaguirre, C., Carotid body grafts induce chemosensitivity in muscle nerve fibers, Brain Research, 270 (1983) 77-92. 31 Morris, J. H., Hudson. A. R. and Weddell, G., A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. II. The developing of the regenerating unit, Z. Zellforsch., 124 (1972) 103130. 32 Morris, J. H., Hudson, A. R. and Weddell, G., A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. IV. Changes in fascicular microtopography perineurium and endoneurial fibroblasts. Z. Zellforsch., 124 (1972) 165-203. 33 Nishi, K., Okajima, Y.. Ito, H. and Sugahara, K., Alteration of chemoreceptor responses and structural features of ischemic carotid body of the cat, Jap. J. Physiol., 31 ( 1981 ) 677-694. 34 Paintal, A. S., Some considerations relating to studies on chemoreceptor responses. In: R. W. Torrance (Ed.), Arterial Chemoreceptors, Oxford, Blackwell, 1968, pp. 253261.
35 Sampson, S. R., Aminoff, M. J., Jaffe, R. A. and Vidruk, E. H., Analysis of inhibitory effect ofdopamine on carotid body chemoreceptors in cats, A mer. J. Physiol., 230 (1976) 1494-1498. 36 Scadding, J. W., Development of ongoing activity, mechanosensitivity, and adrenaline sensitivity in several peripheral nerve axons, Exp. Neurol., 73 ( 1981 ) 345- 364. 37 Smith, P. G. and Mills, E., Autoradiographic identification of the termination of petrosal ganglion neurons in the cat carotid body, Brain Research, 113 (1976) 174-178. 38 Smith, P. G. and Mills, E., Physiological and ultrastructural observations on regenerated carotid sinus nerves after removal of the carotid bodies in cats, Neuroscience, 4 (1979) 2009- 2020. 39 Verna, A., Roumy, M. and Leitner, L.-M., Loss of chemoreceptive properties of the rabbit carotid body after destruction of the glomus cells, Brain Research, 100 (1975) 13-23. 40 Wharton, J., Polak, J. M., Pearse, A. G. E., McGregor, G. P., Bryant, M. G., Bloom, S. R., Emson, P. C., Bisgard, G. E. and Will, J. A., Enkephalin-, VIP-, and substance Plike immunoreactivity in the carotid body, Nature (Lond.), 284 (1980) 26%271. 41 Zapata, P., Effects of dopamine on carotid chemo- and baroreceptors in vitro, J. Physiol. (Lond), 244 (1975) 235-251. 42 Zapata, P., Hess, A., Bliss, E. L. and Eyzaguirre, C., Chemical, electron microscopic and physiological observations on the role ofcatecholamines in the carotid body, Brain Research, 14 (1969) 473-488. 43 Zapata, P., Stensaas, L. J. and Eyzaguirre, C., Axon regeneration following a lesion of the carotid nerve: electrophysiological and ultrastructural observations, Brain Research, 113 (1976) 235-253.
63
Elsevier
Effects of Ischemia on the Function and Structure of the Cat Carotid Body L. MONTI-BLOCH*, L. J. STENSAAS and C. EYZAGUIRRE**
Department of Physiology, University of Utah, School of Medicine, Research Park, Salt Lake City, UT84108 (U.S.A.) (Accepted November 30th, 1982)
Key words: ischemia - carotid body - chemosensory discharge
Interrupting the blood supply to the carotid body by ligating arteries of its vascular peduncle altered the chemoreceptive properties of the carotid nerve and produced structural changes in parenchymal (glomus and sustentacular) cells. The onset ofischemia was marked by an increase in the discharge of both A (myelinated) and C (unmyelinated) sensory fibers followed by depression and finally by receptor silence. The discharge of A-fibers disappeared after 30-50 min and that of C-fibers after 60-90 min. During ischemia of 15-60 min duration the threshold to pharmacological (NaCN, ACh) and 'natural' (hypoxic) stimuli progressively increased and was accompanied by reversible changes in the structure of parenchymal cells and nerve endings, lschemia for 2 h or longer produced irreversible functional damage and disappearance of glomus and sustentacular cells from the carotid body. Following ischemic injury, nerve fibers regenerated and all responded to mechanical stimuli but only a few were stimulated by natural or pharmacological agents. Thus, parenchymal cells of the carotid body appear to be most important in transduction by allowing sensory fibers to respond to chemical stimuli. INTRODUCTION
The carotid body is a complex sensory receptor formed by myelinated (A) and unmyelinated (C) sensory fibers j3 which innervate the glomus cells in the organ. The glomus cell-nerve ending junction is enveloped by sustentacular cell processes. For a number of years it has been assumed that integrity of the whole receptor complex (cells and nerve fibers) is essential in the transduction process This view has been supported by several investigators who have indicated that sensory fibers of the carotid nerve are incapable of normal chemoreception without the presence of carotid body cells7,33,38.39,43.Others have suggested the opposite, namely, that the parenchymal cells are not essential components of this process3,24. 27.
In view of these discrepancies we sought to clarify the role of carotid body cells in transduction by using acute and chronic ischemia to destroy them. These experiments revealed that total short-term ischemia (60 min or less) induced
functional and morphological alterations that recovered after restoring circulation or placing the preparations in vitro. However, arterial occlusion for 2 h or longer led to irreversible functional and morphological damage to the chemoreceptors whose response to natural and pharmacological stimuli disappeared after ischemia. Our results thus agree with those of others who eliminated the carotid body by cryodestruction39 or who removed it surgically in the cat38 or human2~. It appears, therefore, that carotid body parenchymal cells are important in chemotransduction. They seem to condition the afferent fibers to respond to chemical stimulation. Some preliminary results have been published elsewhere29. MATERIALS AND METHODS
Surgical procedures for the production of carotid body ischemia Experiments were performed in 43 adult cats anesthetized with sodium pentobarbitone (40
* Present address: Departamento de Fisiologia, Facultad de Medicina, Universidad de la Repfiblica, Montevideo, Uruguay, ** To whom correspondence should be addressed. 0006-8993/83/$03.00 ,,~ 1983 Elsevier Science Publishers B.V.
64 m g / k g i.p.) and heparinized (500 U/kg, iv.). The right carotid body was identified and the vascular peduncle which provides the arterial supply to the organ was exposed. A ligature consisting of 6-0 silk thread was placed around the vascular peduncle; ligatures were also situated on the occipital and the ascending pharyngeal arteries close to the carotid body (Fig. 1, arrows). The veins draining the organ were left intact. Four groups of experiments were performed: (1) in 22 cats, acute ischemia (15 min to 6 h) was produced and the ligatures were left in place; (2) in 9 animals, acute ischemia was followed by short-term recovery (30 min to 4 h), by removal
oca e~
cca Fig. 1. Schematic drawing of carotid body region and ligated blood vessels (arrows): cca, common carotid artery; eca, external carotid artery; ica, internal carotid artery; oca, occipital artery; aph, ascending pharyngeal artery; cb, carotid body; sn, sinus (carotid) nerve; scg, superior cervical sympathetic ganglion; cv, carotid body vein.
of the ligatures or placing the carotid body in vitro and superfusing it with oxygenated saline; (3) in 6 preparations, acute ischemia was followed by prolonged recovery, by removing the ligatures for 18-48 h; (4) in 6 cats, chronic ischemia (12 h to 10 months) was produced by leaving the ligatures in place. Operations were performed under aseptic conditions when the intended survival period was 18 h or longer.
Electrophysiology In situ recordings were made from both normal and ischemic carotid body preparations. The ganglio-glomerular nerves were cut and baroreceptor fibers were crushed around the carotid sinuses to eliminate mechanoreceptor activity ~2. The carotid nerve was surgically isolated, sectioned close to the glossopharyngeal nerve, and protected from drying by warm mineral oil. Action potentials were recorded from the nerve with platinum-iridium electrodes connected to a conventional AC recording system. Nerve discharges were stored on FM tape (frequency response, DC to 1250 Hz) for analysis and photography. Frequency changes of sensory units, selected with a window discriminator, were measured with a counter and printer system and fed to a digital-analog converter for display on a chart recorder or oscilloscope. Conduction velocity of afferent fibers in small filaments containing one or few active units was estimated by utilizing a double set of recording electrodes (5 m m separation) to measure differences in arrival time of the same action potentials ~3. This method gives only an approximate estimate of conduction velocity in different fiber groups, because of the short length of nerve between the pairs of recording electrodes 34. Chemosensory responses to systemic or 'topical' application of natural and pharmacological stimuli or inhibitors were studied. Hypoxia was produced by inhalation of 100% N2 or by gently blowing this gas over the carotid body for 1-2 min, using a 23-gauge needle placed close to the organ and connected to the gas reservoir. For hyperoxia the same procedures were used for 100% 02. Nicotine bitartrate (K and K), NaCN (Merck) and dopamine HC1-DA (Sigma) dis-
65 solved in modified Tyrode's solution were either injected into the femoral vein or applied directly (using a microdispenser) to the carotid body in volumes of 5 /~1. Acetylcholine chloride-ACh (Sigma) was always applied directly. The topically applied drugs were removed by repeated rinsing of the preparation with 0.25 ml of warm saline. In vitro recordings were obtained from 7 ischemic preparations. The carotid bodies and their own nerves were dissected out and placed in a chamber through which flowed modified Tyrode's solution (pH 7.43) at 36 °C. Gases and drugs were delivered by using methods previously reported s,~l.~,_2s. Electron microscopy Tissue was prepared for ultrastructural analysis with a fixative containing 0.1 M phosphate buffer (pH 7.4), 1% paraformaldehyde and 1% glutaraldehyde. Carotid bodies with ligatures situated on the vascular peduncle were removed and fixed by immersion for 8 12 h. Preparations with an intact vascular supply were perfused at a pressure of 100 torr for 10 min, the carotid bodies removed and placed in cold fixative for 8- 12 h. All tissue was rinsed in 2.5% NaC1, postfixed in 2% osmic acid in 0.1 M phosphate buffer for 2 h, dehydrated in a graded series of alcohol and embedded in Epon. Semithin sections were cut through the mid-portion of the carotid body. The blocks were retrimmed; ultrathin sections were cut, mounted on one-hole (1 mm x 2 mm) grids on Formvar film and stained with uranyl acetate and lead citrate. Reactive changes in intralobular and extralobular constituents of the carotid body were determined electron microscopically by systematic examination of single ultrathin sections through the mid-portion of the carotid body. The evaluation of each constituent was facilitated by the use of one-hole grids which afforded an unobstructed view of the entire section. Twenty-five glomus cells in which the nucleus was visible were selected for a detailed examination of reactive changes. The axon terminals, sustentacular cells and unmyelinated intralobular axons associated with these cells were subsequently inspected. Finally, myelinated and unmyelinated
axons between the lobules containing the glomus cells were systematically examined for reactive changes. Information was then tabulated: (0) indicated absence or necrosis of a given element, ( - ) few reactive constituents, ( - - ) many reactive constituents, ( + ) normal incidence of a given element, and ( + + ) supra-normal incidence. RESULTS
The effectiveness of the ligatures in producing ischemia was evaluated morphologically. Of 43 carotid bodies examined with the electron microscope, 22 (51%) showed unequivocal signs of total ischemia while the rest (49%) showed that ischemia was incomplete. The reasons for such variability are unknown since an effort was made to tie all visible arterial vessels entering the carotid body. On two occasions, both arterial and venous vessels were tied, resulting in faster and more profound changes than when only the arteries were ligated. This combined procedure was not employed routinely because of the possibility of damaging the carotid nerve. Physiological observations A cute ischemia Ligature of the carotid body vascular peduncle resulted in an increase in frequency of the chemosensory discharge, which reached a peak in about 5 min; this was followed by a progressive decrease, until complete silence was attained at nearly 90 min (Fig. 2). When sensory units with different spike amplitudes were analyzed through the window discriminator, responses with different temporal patterns were observed. Ischemia induced a significantly (P 0.01) larger increase in frequency for sensory units with larger spikes (A), than for those with small spikes (B). Also, the fibers with larger spikes (A) ceased firing after 3(L50 min of continued ischemia, whereas those with small spikes (B) did so 60-90 min after applying the ligature. Conduction velocity measurements showed that the low amplitude action potentials corresponded to sensory fibers conducting at 0.5-0.9
66
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Fig. 2. Frequency of spontaneous chemosensory discharges recorded from carotid nerve after tying carotid body vascular peduncle. Filled circles and triangles, mean frequency ( ± S.E.) during 60 s recordings. A and B, two different populations ofchemosensory fibers with large and small spikes, respectively. C, control discharge levels.
m/s, while the larger spikes occurred in sensory units conducting at 5-10 m/s. These conduction velocities are comprised within the ranges reported for A and C carotid nerve chemosensory fibers from normal carotid body preparations 13. When hypoxic stimulation (100% N2) was topically applied (Fig. 3) the discharge of the normal carotid nerve increased markedly (A). These fibers became almost silent after 60 min of ischemia (B), but another topical application of 100% N 2 w a s still capable of inducing some receptor stimulation (C). The responses to topical application of stimulants such as ACh 25-100 #g or NaCN 10/~g and the inhibitor DA 100/~g35m were depressed after 15-30 min ischemia and absent when the preparation was ischemic for 60 min or more The fact that DA failed to depress discharges when ap-
plied to 60 min ischemic preparations may have been due to the very low basal discharge (Fig. 2) with little room for further depression Fig. 4 illustrates the dose-dependent responses to ACh of normal preparations (open circles) and preparations subjected to ischemia. Relatively short ischemia (15 min) depressed all responses to ACh, whereas only a minimal response to the largest doses of ACh (100 ~g) was elicited after 30 min ofischemia; all responses to the drug were abolished after 60 min of ischemia. During ischemia there were variations in the response of different preparations to each type of stimulus. At times, the response to ACh was eliminated after 30 min ofischemia while that to 100% N 2 still occurred. In other instances the opposite was true.
67
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Fig. 3. A: carotid nerve discharges during topical 100%N 2 to normal carotid body. B: spontaneous chemosensoryactivity of same preparation after 1 h ischemia. C: 100%N 2 applied after I h ischemia.
Results just presented refer to preparations in which, morphologically, complete ischemia was established. Preparations in which ischemia was incomplete were still able to respond to inhaled N 2 and to systemic injections of N a C N , nicotine and DA for several hours after ligature of the vascular peduncle. An interesting feature occasionally observed was that a preparation partially ischemic for 45 60 min apparently became hyper-reactive to different stimuli. For instance,
100% N2 inhalation induced a peak/baseline discharge ratio of 1.1 (dF = +38.3 Hz) in the normal preparation with a mean baseline discharge of 156 Hz. Partial ischemia produced a great initial increase in chemosensory discharges, but after 60 min, the mean baseline frequency decreased to 28.1 Hz and during 100% N 2 inhalation the peak/baseline ratio increased to 13.1 (dF = +87.3 Hz). Similar results were obtained in the same preparation during topical applications of ACh 25/~g. Before ischemia the peak/baseline ratio was 1.5 (dF = 85.7 Hz), which increased to 2 2 (dF = 340 Hz) after 60 min of ischemia. The reasons for these differences are unknown and require further investigation Chronic ischemia Chronic ischemia was produced by ligation of the vascular peduncle for periods ranging from 12 h to 10 months. The carotid body appeared
68
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Fig. 5. Carotid nerve discharges from long-term ischemic carotid bodies. A, small action potentials from 2-day ischemic glomus; B, small and larger spikes in 7-day ischemic preparation: C, local tapping of 7-day ischemic glomus induces burst of action potentials: D, spontaneous discharges from I month ischemiccarotid body. swollen after 2 days, but could not be identified under the stereomicroscope (6-25 x ) after 7 days. A great reduction in spontaneous activity was observed after 2 and 7 days of complete ischemia, and consisted of very small action potentials and a few larger spikes (Fig. 5A and B). Topical application of 100% N2, ACh 100 ~g, nicotine 64 ~g, NaCN 10 /~g and DA 100 /xg usually did not change the spontaneous discharge frequency. Mechanical stimulation of the tissue attached to the carotid nerve in the 7-day preparation gave rise to short bursts of activity (Fig. 5C). Ischemia for 1 month resulted in a very marked reduction in spontaneous activity, consisting of both very small and somewhat larger spikes (Fig. 5D). The experiment illustrated in Fig. 5D was analyzed to compare nerve activity in the ischemic and normal carotid body. The carotid nerve of this 1-month ischemic preparation had a basal discharge of 1.4 _+0.3 Hz (mean + S.E.) as opposed to 157 + 2.6 Hz on the normal side. This indicates that the operated side had an activity which was 0.09% of normal. Stimulation by inhalation of 100% N, showed that the reacti-
vity of the few units remaining in the operated side was not significantly different from the normal preparation. In fact, the mean peak/baseline discharge ratio was 3.76 on the operated side vs 1.21 on the normal preparation (P 0.2). When 100% 02 was inhaled these ratios were 0.88 and 0.54 respectively (P ~ 0.001) indicating that 100% 02 was more effective on the normal preparation. Differences between the effects o f N 2 and 02 tests may have been due to the very different baseline discharge in both cases9. Ultrastructural control of this ischemic preparation revealed an absence of both glomus cells and sensory nerve terminals, but an abundance of unmyelinated axons. It is possible that during this long ischemic period regenerating axons may have innervated some of the numerous miniglomera located near the carotid body, which continued to respond to the usual chemosensory stimuli 26. Alternatively, a few regenerating chemosensory fibers may have been able to respond directly to the aforementioned stimuli. In fact, regenerating mammalian fibers in other systems show spontaneous activity which can be modified by substances such as adrenaline 5,6,36.
69 Chronic partial ischemia (1 day-10 months) usually induced a depressed response to different stimuli. For example, one partially ischemic carotid body kept for 10 months was tested with topically applied 100% N2, N a C N 10 /zg, ACh 100 ~g and DA 100/~g. The peak/baseline ratios obtained from the operated side were 52% for N2, 32% for NaCN, 14% for ACh and 40% for DA of the ratios recorded from the contralateral normal cartid body. Similar reductions were observed when dF values were compared. These observations indicate that a prolonged ischemic insult may produce permanent physiological damage to the glomus-sustentacular complex even though it may survive as a morphological entity. In all probability nerve fibers injured during this process had ample time to regenerate.
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Restoration offunction after acute ischemia Short-term recovery. Preparations totally ischemic for 15 min to 2 h either had the vascular ligatures removed or were placed in vitro to study possible recovery of chemoreceptor function. Results were similar in both experimental situations. After a 30-min ischemic period the responses to 100% N: or NaCN 5 /~g were markedly reduced and recovered only partially either in vitro or after restoring circulation. The depressed responses to ACh 100 ~g or DA 40 ~tg topically applied were not restored. Ischemia for 60 min resulted in a significantly reduced response to 100% N 2. Placing the preparation in vitro or restoring circulation for 1 h induced partial recovery only in some preparations (Fig. 6). As shown before, applications of
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70 NaCN 10/.% ACh 100 ~g and DA 100 #g were ineffective in changing the sensory discharge frequency after 60 min ischemia. These effects failed to recover in vitro or after removing the ligatures for 1 h. Long-term recovery. Spontaneous activity consisting only of small spikes was present in preparations allowed to recover for 2 days following 60 min ofischemia. These fibers had conduction velocities of 0.4-0.7 m/s corresponding to he slower fibers of the intact carotid nerve. The frequency of discharges from the whole nerve was well below the discharge level of the contralateral normal carotid nerve. Natural or pharmacological stimuli did not change the activity of these preparations. However, mechanical stimulation of the carotid bifurcation induced volleys of small action potentials in fibers that were not spontaneously active. Inspection of the region showed either no visible carotid body or one which was quite small. TABLE
A 1 h ischemic preparation allowed to recover for 30 days showed no identifiable carotid body within the carotid bifurcation. The nerve contained few spontaneously active fibers producing small action potentials and a few spikes of higher amplitude with conduction velocities of 0.5-1.5 m/s. Applications of 100% N2, ACh 100 /~g and NaCN 10/tg were without effect. However, mechanical stimulation of the area produced short volleys of action potentials. These results indicate that chemoreceptor function altered by an ischemic insult can be partially restored if ischemia lasted 60 min or less. More prolonged periods of ischemia (2 h or longer) induced permanent damage to the chemoreceptor complex.
Morphological observations Reactive and degenerative changes following ischemic periods up to 12 h involved a progres-
I
Morphological changes in ischemic carotid bodies +, Normal; + +, supranormal number; -, few reactive/many normal; --,
Periods
Intralobular constituents
Ischemic
Recovery
Glomus cells
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m a n y r e a c t i v e / f e w n o r m a l ; O, absent or necrotic.
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71 sive and time-dependent sequence. Table I summarizes the morphological findings concerning reactive changes in the parenchymal components of lobules and in axons of the extra-lobular compartment (stroma). The sequence of ultrastructural changes involved: axon terminal detachment (Fig. 8), degeneration of glomus cells (Fig. 10), retrograde degeneration of extralobular axons (Fig. 11), disappearance of glomus and sustentacular cells, and regeneration of axons into the depopulated connective tissue of the carotid body (Fig. 12). Reactive ultrastructural changes developed rapidly following short periods of ischemia. They were evident in all lobular constituents, and intially involved alterations in their appositional relations. Cytological changes in glomus cells appeared before alterations were detected in sustentacular cells, Schwann cells or axons (Figs. 7 9). The fact that changes followingshort-term ischemia with no recovery were vir-
Fig. 7. Fifteen min ischemia, 1 h recovery. Pror~inent extracellular spaces (asterisks) occur between glomus (GC) and sustentacular cell processes (arrows). Spaces minimally affect axon terminals which remain apposed to glomus cells in 'edematous' lobule. 7375 x .
tually identical to those of ischemia followed by short periods of recovery (1-4 h) indicated that such cytological alterations occurred rapidly. Changes occurring up to 2 h of ischemia were proportional to the duration of the ischemic period. The most conspicuous were those affecting the cytoplasm of glomus cells, and those involving detachment of sustentacular processes; such changes were apparent after only 15 min of ischemia and 1 h recovery (Fig. 7). The spaces which developed at the sites of detachment gave the lobules an 'edematous' appearance. In general, glomus cells in such lobules displayed a reduced incidence of granular vesicles and a cytoplasm markedly lighter than normal. However, in lobules lacking intralobular spaces some cells had a cytoplasm darker than normal; such cells also had an increased incidence of granular vesicles and were abnormal in size and shape. Major alterations in synaptic relations occurred after 30 min ischemia and 1 h recovery. They involved a substantial reduction in the area of apposition between nerve terminals and glomus cells in certain lobules. As shown in Fig.
Fig. 8. Thirty rain ischemia, 1 h recovery. Early stage ofaxon terminal (AT) detachment involves intercalation ofsustentacular cell processes (arrows) between axon and glomus cell (GC). Note unusual profusion ofsustentacular cell processes within intralobular spaces (circled). 6450 x .
72 8, thin lamellar processes of sustentacular cells were interposed between glomus cells and axonal enlargements identified as calyciform terminals. The process of axon terminal detachment through the interposition of a single lamella of a sustentacular cell was common in the non-edematous lobules. However, there were a few instances of multiple lamellae of sustentacular cells separating axon terminals from glomus cells with dark cytoplasm. Also, edematous spaces separated a few axon terminals and glomus cells with light cytoplasm. Ischemia for 1 h appeared to be at or near threshold for inducing death of parenchymal cells since some necrotic glomus cells were present in 1-h preparations following recovery times of 24 or 48 h. These carotid bodies also showed a profound reduction in the number of terminals apposed to glomus cells (Fig. 9) and a reduced incidence of intralobular unmyelinated axons. However, there was no reduction in the number ofmyelinated or unmyelinated axons in the stroma and no clear signs ofaxonal degeneration, although the cytoplasm of all axons was darker than normal. Reduction in the number and extension of synapses in the absence of Wal-
Fig. 9. One h ischemia, no recovery. Extensive detachment of axon terminals (AT) occurs by intercalation of multiple reactive sustentacular cell processes (arrows) between axon and glomus cell (GC). 11,100 ×.
~, ~
Fig. 10. Two h ischemia, no recovery. Necrotic glomus cell (GC) undergoing lysis, phagocytosis (arrows) and lysosomal disintegration within sustentacular cells (white asterisks). Note unapposed nerve profiles resembling axonal termirials (AT) and intralobular unmyelinated axons (black asterisks). 6450 ×.
lerian degeneration appeared to result from terminal detachment followed by withdrawal of the axon from the lobule, possibly involving retraction into interlobular myelinated axons within the stroma. Carotid bodies subjected to 2 h of ischemia without a recovery period contained many necrotic glomus cells. The 2-h period was above threshold for inducing cell necrosis. Both 1- and 2-h ischemic preparations with 1 h of recovery displayed extensive reactive changes in the cellular constituents of all lobules. Intralobular axons having the cytological characteristics of terminals but lacking contact with glomus cells were common. All were surrouncaed by multiple lamellate processes arising from reactive sustentacular cells. Cell necrosis after 2 or more hours of ischemia involved lysis of light glomus cells and pyknosis of the dark cells. Fragmentation and disintegration of a glomus cell induced an
73 immediate phagocytic response in nearby sustentacular cells (Fig. 10). Preparations recovering for more than 1 h after 1 and 2 h ofischemia contained few axon terminals and few intralobular unmyelinated axons, but the incidence of extralobular myelinated axons was normal. Thus, the loss of intralobular axons through carotid body ischemia appears to be related to reactive changes leading to the disintegration and disappearance of glomus cells. Reactive changes in extralobular myelinated axons included shrinkage and the development of dark cytoplasm with irregular cisternae. Dense-core vesicles were somewhat more numerous in the axons of nerves which had been cut to permit recording of action potentials than in intact nerves. Parenchymal elements in various stages of disintegration and removal were present in 2-h ischemic preparations recovering for 12 h. Disappearance of the lobular constituents left connective tissue in which there were reactive nerves, granular leucocytes, fibroblasts, and reactive sinusoid capillaries. Loss of axoplasm was characteristic of interlo-
Fig. 11. Twelve h ischemia, no recovery. Interlobular unmyelinated (U) and myelinated axons (M) in association with necrotic glomus cell (GC). 6340 x .
Fig. 12. Seven day ischemia, no recovery. Axons (A) resembling regenerating axon sprouts occur in the carotid body in association with Schwann cells. 11,100x.
bular myelinated axons following 12 h of ischemia; however, many axons survived in spite of a persisting impediment to circulation (Fig. 11). Axons having the fewest reactive changes were those invested by an intact and well developed perineurial sheath. Regenerating unmyelinated axons were present in the carotid body of animals which survived for 7 and 16 days after 2 or more hours of ischemia. They appeared to originate from myelinated axons which had undergone few retrograde changes. The cytoplasmic characteristics of regenerating axons in the carotid body stroma were identical to those of the regenerating peripheral nerve (Fig. 12). However, perineurial components were either absent or lacking around such axons and there was little or no 'compartmentation '3~-32. In all cases, axons occurred in association with Schwann cells. Persistence of unmyelinated axons resembling axon sprouts together with numerous myelinated axons in animals surviving 30 days or more indicate both continuing axonal growth and remyelination in the absence of glomus cells. The unmyelinated axons were observed to have neither an external configuration characteristic of normal intralobular axons nor to contain the aggregates of vesicles typical of terminals in the normal carotid body.
74 DISCUSSION
Heymans and Bouckaert 2° and Alvarez-Buylla ~ altered the chemoreceptor properties of the carotid body by occluding the circulation within the organ using injections of Lycopodium spores or talcum powder. Alvarez-Buella ~reported that such occlusion induced an increased carotid nerve chemosensory discharge lasting 15 min, followed by disappearance of spontaneous chemosensory activity and elimination of chemoreceptor responses, while leaving barosensory activity intact. Nishi et al. 33 surgically occluded blood vessels to the carotid body and found similar changes. They observed recovery after restoring circulation to the preparation, provided that the ischemic period lasted no longer than 60 min. Our results are in essential agreement with all of the earlier studies. Whether the reduction in chemosensory function was due primarily to alterations in glomus cells or to changes in the sensory nerve terminals could not be determined from the ultrastructural changes following short-term ischemia (less than 60 min), since both were altered. Cytological changes of sensory nerve terminals were generally less conspicuous than those of glomus cells; but since the morphological integrity of both elements was compromised, it suggests that both may have been involved. The morphological changes correlated well with the physiological findings following both long-term ischemia (up to 30 days) and ischemia of 60 min or more with long-term recovery. In these cases glomus cells became necrotic and disappeared while the nerve regenerated. Our finding that the great majority of sensory fibers in such preparations lacked chemoreceptive properties contradicts the suggestion of others that glomus cells are not required for chemotransduction24,27. Present results are in accord with the findings ofVerna et al? 9 who destroyed parenchymal cells of the carotid body by freezing and allowed time for the nerve to regenerate: such preparations showed no chemosensitivity when cryodestruction was complete. Our conclusions also agree with the findings of Smith and Mills 38 who removed both carotid bodies
and showed no carotid nerve chemoreceptor activity in animals tested several months later. Similarly, studies in humans following bilateral removal of the carotid body to alleviate asthma showed severe impairment of the ventilatory hypoxic response 2j. Therefore, most of the available evidence indicates that glomus cells are necessary for chemotransduction and that regenerated or regenerating carotid nerve fibers cannot perform this function alone. This agrees with the experiments of Zapata et al. 43, who crushed the carotid nerve and found no chemoreception until the regenerating fibers became apposed to carotid body parenchymal cells. Our experiments further suggest that peripheral, preneural elements (glomus cells in this case) are capable of 'conditioning' the sensory nerve fibers to respond to particular sensory stimuli. Such changes in the functional properties of axons may involve transfer of a chemical signal from the glomus cell to the perikaryon of the sensory neuron via retrograde axonal transport. However, no detailed analysis of changes in centripetal transport of materials before and after removal of glomus cells has been performed. It is known that the carotid nerve can transport horseradish peroxidase from the periphery to the sensory (petrosal) ganglion and central processes2,4,23 as it can transport radiolabeled amino acids from this ganglion to the carotid b o d f 4-37.Thus, while there is no direct evidence that retrograde transport may influence petrosal sensory neurons and render its endings chemosensitive, differences between the electrical properties of chemo- and barosensory ganglion cells indicate this possibilitf 6. If a peripheral contribution is required to condition the behavior of sensory neurons, the situation may be similar to that of mammalian motoneurons some of whose properties are dependent on the muscle cells which they innervate 15. Despite lack of information as to the means by which carotid body parenchymal cells influence sensory neurons, it is known that ACh and catecholamines are contained in these cells and are released during stimulation ~°,~2-~8-19.More recently, it has been found that glomus cells also contain enkephalins 25,4° and that substance P is
75 present in carotid nerve fibers 22 and petrosal ganglion cells 17. These chemicals, plus others still not identified, may be involved in trophic interactions between glomus cells and sensory neurons and may induce the carotid nerve afferents to behave as chemoreceptor elements. This possibility is explored further in the following paper where the carotid body was grafted into a muscle and innervated with fibers from the muscle nerve3°.
ACKNOWLEDGEMENTS
We wish to express our appreciation to Dr. P. Zapata for reading this manuscript and providing valuable suggestions. We are also grateful to Messrs. P. Lenoir, J. Fisher and B. Evans for technical assistance. Mrs. Carolina Zapata helped in the preparation of this paper and illustrations. Work sponsored by Grants NS-05666 and NS-07938 from the U.S. Public Health Service.
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