and carotid sinus (sino-aortic) denervation (S-A D) at different postnatal ages in the ... involving dissection of the carotid sheath with exposure of the carotid sinus.
Pergamon Press
Life Sciences, Vol. 34, pp. 489-496 Printed in the U.S.A.
LETHAL RESPIRATORY DISTURBANCE IN NEONATAL RATS AFTER ARTERIAL CHEMORHCEPTOR DENERVATION Myron A. Hofer Depts. of Psychiatry and Neuroscience Albert Einstein College of Medicine at Montefiore Medical Center Iii East 210th Street Bronx, New York 10467 (Received in final form November 17, 1984)
summary The role of afferent feedback from arterial chemoreceptors in the maintenance of rhythmic respiration during early development was studied by section of carotid sinus and aortic nerves of rat pups at different ages from 3 days to 3 ~ e k s postnatally. This deafferentation produced a severe, episodic respiratory disturbance, limited to pups younger than 21 days and associated with mortality rates near 50% during the 2 weeks following surgery. These findings may have implications for the role of peripheral chemoreceptors in the periodic apneas of premature infants and in the Sudden Infant Death Syndrome. Premature infants with frequent episodes of apnea and atypical "periodic" respiration have raised questions about how rhythmic respiration is maintained during early development (i). Pathological changes in the carotid bodies of victims of sudden infant death have suggested a crucial role for peripheral chemoreception (2). On the other hand, chemoreceptor deafferentation in neonatal animals has generally failed to disrupt rhythmic respiration (3). These experimental studies are not conclusive, however, because they have been limited either to the anesthetized state or to section of only carotid sinus afferents or to a precocial species, such as the sheep, which is born more mature, neurobehaviorally, than the human. We studied the effects of aortic and carotid sinus (sino-aortic) denervation (S-A D) at different postnatal ages in the infant rat, an altricial species born in a relatively immature state. Methods Rats of a wistar-derived strain (Marland Farms), born and raised in our laboratory, were housed in terraria (40 x 20 x 24 ~n) under conditions of regulated temperature and humidity and a reversed 12:12 light-dark cycle. Two to three days prior to study, pups were lightly anesthetized with ether and implanted Lwith chronic subcutaneous silver wire electrodes for recording thoraco-abdominal respiratory movement patterns by impedance pneumography and cardiac rates by cardiotachometer on a Grass Model 7 polygraph (4). Surgery was performed under 12-25 x magnification using a high resolution stereoscopic operating microscope (Wild Model 5A) and fiber optic illtlnination. The carotid sinus nerve and aortic depressor nerve fibers were identified (5) and cut. Control operations consisted of dissection down to 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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the carotid sheath and, in the younger groups, a more extensive procedure, involving dissection of the carotid sheath with exposure of the carotid sinus and aortic fibers in an identical manner to the denervation operations. The effectiveness of the-denervation procedure was assessed by measuring the arterial cln~noreceptor-mediated respiratory response to brief hypoxia and hyperoxia, under urethane anesthesia. Pups were placed on their backs and given 100% nitrogen, 100% oxygen or air (as a control) delivered at 500 ml/min through a plastic hose placed loosely over the snout. Severe hypoxiawas rapidly produced by 15 sec. of nitrogen, and hyperoxia by 30 sec. of pure oxygen; 1-2 mins. were allowed between inhalation tests. The respiratory rates for the last 10 breaths of each t e s t w e r e c o m p a r e d w i t h the 10 breaths prior to experimental inhalation, to obtain respiratory rate change figures. Deafferentation and control surgerywere performed under ether anesthesia on 3-5, 8-10, 14-16 and 20-22 day old pups between 9 a.m. and noon. The pups were replaced in their home cage with littermates after recovery from the ether anesthesia. At 4:30 p.m., 4-6h after surgery, 5 min. recordings were made by attaching lightweight leads to the chronic electrodes of sleeping pups in their home cage nest. Following physiological recordings, the mothers were returned to their litters overnight. A second set of 5 min. recordings and tail cuff blood pressures were taken 24h after surgery and a third set of recordings was made l week later. All pups were followed for two weeks with weights taken at 1 day, 1 week and 2 weeks fbllowing surgery. The impedance pneumograph recordings from the longitudinal study were analyzed according to the categories shown in Fig. I. Irregular respiration, the pattern typical for REM sleep in the infant rat (6), consists of alternating fast and slow periods, with maintenance of the usual sine wave pattern of inspiration and expiration (Fig. la). Respiration was identified as atypical when the usual sine wave pattern of linked phases was replaced, for at least 5 sec., by an arrhytlmaic series of low amplitude waveforms with frequent short pauses between inspiratory efforts and occasional high amplitude gasps (Fig ic,d). Apneas were defined as periods with little or no respiratory movement, lasting for longer than 3 normal breaths (2 sec.). Movement artifact prevented scoring of respiration during activity. The ntmlber and duration of periods of atypical respiration and apnea were measured and the total time spent in these respiratory patterns during the 10 min. recordings were obtained for each pup. Inter-rater reliability across subjects was 0.93 for atypical respiration time and 0.96 for apnea time. In addition, we obtained mean (regular) respiratory rates and mean cardiac rates during each respiratory pattern on all subjects. Results Spontaneous Respiratory Patterns: The respiratory pattern was not visibly affected by the denervation surgery under anesthesia, but during recovery, younger pups began to have short periods of grossly irregular, low amplitude respiration with pauses and occasional gasps. Cyanosis became clearly evident over their hairless bodies. Impedance pnetmxlgraph recordings of these patterns are shown in the bottom two examples in Fig. i, where they are clearly distinguishable because of the loss of the sine wave pattern, the frequent short pauses and the variability of waveform from one breath to the next.
Vol. 34, No. 5, 1 9 8 4
Sino-aortic Denervation in Infant Rats
491
c.
d '
j
I
[ ....
i ....
I ....
I ....
J
,.I
i
Seconds Fig. 1 Patterns of respiration, recorded by the transthoracic impedance method, from unanesthetized, unrestrained infant rats with littermates in home cage. A, regular respiration, characteristic of the slow wave sleep state. B, irregular respiration, characteristic of REM and of quiet awake states. C, short episode of atypical respiration, with brief apneic spell (underlined) in 9 day old pup, 24h after sino-aortic denervation. D, excerpt from longer and more severe episode of atypical respiration recorded fram a 3-5 day old pup, 4 hours after recovery from sino-aortic denervation. This pup was found dead the following morning.
Respiratory Responses to Hypoxia and Hyperoxia: In order to ascertain whether our microsurgical procedure was in fact denervating the arterial chemoreceptors, a procedure was utilized (see Methods) that reliably elicited respiratory reflexes to hypoxia and hyperoxia known to occur in neonatal animals and to be dependent on intact arterial cheaoreceptors (7). In the acute studies, 8-11 day old animals were anesthetized with urethane, tested for respiratory responses, then subjected to denervation or control operation and finally retested with nitrogen and oxygen inhalation. In the longer term studies, another series of 8-10 day old animals were operated under ether, and replaced with their mothers for one week. At the end of this time, the surviving denervated pups (S-A D) and the controls were anesthetized with urethane and tested as in the acute studies. The results are presented in Table I.
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Table I Respiratory Responses to Inhaled Gases
Pretest Rate cyc/min
Oxygen cyc/min
Nitrogen cyc/min
Air cyc/min
Day of Surgery - Age 8-11 Days Control
4
S-A D
4
114 (8) 90(9) 1
-23 (5) **
+39 (5) **
-3 (4)
+ 5(2)1
+ 1(8)1
+5(2)
One Week Post Surgery - Age 15-17 Days Control
7
136 (5)
-14 (4) *
+38 (5) **
+3 (2)
S-A D
6
134 (5)
-13 (7)
+ 7 (4) ,i
+2 (2)
Mean respiratory rate and rate changes (with standard errors of means) in response to hyperoxia induced by 100% oxygen inhalation for 30 sec. and to hypoxia induced by 100% nitrogen inhalation for 15 sec. in urethane anesthetized rat pups. **p
