JOURNALOF APPLIED Vol. 39, No. 1, July
PHYSIOLOGY
1975.
Prinledin
U.S.A.
Site of central chemosensitivity A. H. JANSEN
AND
Perinatal
Laboratory,
University
Research of
Manitoba,
V. CHERNICK Departments of Pediatrics and Physiology, Winnipeg, Manitoba R3E 0 WI, Canada
cannulated via a tracheostomy. Oxygen enriched air was supplied to keep the maternal arterial Po2 above 80 mmHg. Experiments were done on 20 fetal lambs ranging in weight from 2.9 to 5.9 kg. The preparation consisted of an exteriorized fetus with its placental circulation intact. Air inhalation was prevented by quickly capping the snout with a glove filled with warm water. The body temperature was held constant at 39-40°C with dry linen and a heating pad. A fetal femoral arterial catheter was connected to a pressure transducer (Statham P23Db) for continuous monitoring of the systemic blood pressure. The normal phasic blood pressure signal was electronically converted into a mean blood pressure tracing by an Electronics for Medicine recorder. Heart rate was measured by a cardiotachometer (Beckman 9857B) with needle electrodes placed under the skin on either side of the chest. The fetal trachea was cannulated without loss of tracheal fluid and connected to a liquid plethysmograph (14). This apparatus allowed the fetus to ventilate its lungs with liquid and in this respect simulate the intrauterine condition. The ventral surface of the brainstem was reached by ventral craniotomy (6) exposing the medulla oblongata from the pons to the spinal cord and laterally to the tympanic bullae. The dura was opened lengthwise and held back by wire springs. Thus the origins of the cranial nerve roots VI to XII were readily visible beneath the edge of the bone. Loss of CSF could not be prevented and therefore the brain surface was bathed in Mitchell’s mock CSF (18) prewarmed to body temperature. Blood loss from the surgical procedure was minimal. After the craniotomy, but just prior to opening the dura, peripheral chemo- and pressoreceptor function was eliminated in all fetuses studied by bilateral section of the sinus and vagus nerves. T o produce 1ocalized hypoxia, filter paper pledgets, 4 mm in diameter, were saturated in a solution of cyanide, 25 mg/ml of mock CSF, and initially placed unilaterally in a random sequence to discrete areas on the ventral surface of the medulla (Fig. 1). However since these trials were largely negative, in the experiments reported pledgets were applied bilaterally to corresponding sites. By calculation, the amount of cyanide held by two pledgets was approximately 0.45 mg, but since cyanide escapes readily into the air at the pH used, the actual dose reaching the brain surface was probably substantially less. After 2 min the pledgets were removed and the surface of the brain flushed with mock CSF. The mean arterial blood pressure output from the Electronics for Medicine recorder, the
JANSEN, A. H., AND V. CHERNICK. Site of central chemosensitiuity in fetal sheep. J. Appl. Physiol. 39( 1) : l-6. 1975.-The heart rate, blood pressure, and respiratory response to topically applied cyanide on the ventrolateral medullary surface and upper spinal cord was studied on exteriorized sinaortic-denervated fetal lambs under pentobarbital anesthesia. On all sites tested cyanide produced a rapid increase in heart rate and blood pressure (P < 0.05) which was most pronounced from the area adjacent to the nerve roots IX to XI (mean 32 %). Respiratory efforts consisting of l-8 gasps were induced in half the applications to the medulla but never when the pledgets were applied to the spinal cord. The mean delay to response was 43 s (range 13-l 02 s). After cautery of the chemosensitive areas, topical application of cyanide failed to stimulate gasping, whereas intravenous cyanide or cord clamping still produced a vigorous respiratory response. It is concluded that sympathetic stimulation of the heart and blood vessels can originate centrally in response to local histotoxic hypoxia of the ventral medulla and upper spinal cord. Furthermore, it is proposed that in the apneic fetus histotoxic hypoxia of the medulla initiates respiration possibly by stimulating a special gasping mechanism which is separate from the respiratory center responsible for rhythmic breathing after birth. The responsible neurons must be located at least 2 mm beneath the ventral medullary surface.
initiation medulla hypoxia; receptors
in fetal sheep
of fetal respiration; first breath; stimulation of ventral by cyanide; cardiovascular response to cyanide; fetal fetal central chemoreceptor; fetal peripheral chemo-
PREVIOUS EXPERIMENTS demonstrated that cyanide injected into the subarachnoid space of the ventral medulla of exteriorized fetal lambs will induce respiratory efforts and increase blood pressure and heart rate (16). This response was independent of the peripheral chemo- and pressoreceptors indicating a central effect of cyanide. However, the infusion technique used did not permit any conclusions regarding the exact site of action on the ventral surface of the brainstem nor whether the reactive cells were located near the surface or in the area of the respiratory center proper. The present study was undertaken to delineate the site of action of the cyanide more precisely. METHODS
Term pregnant ewes (Dorset-Suffolk) were with intravenous sodium pentobarbital, 15-20 wt, and maintained with additional injections, as required during the experiments. Catheters in a carotid artery and jugular vein and the
anesthetized mg/kg body 2-3 mg/kg, were placed trachea was 1
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
A. H. JANSEN
AND
V. CHERNICK
RESULTS
FIG. 1. Diagram of the ventral side of the medulla of the fetal sheep showing the roots of the cranial nerves V to XII and the areas of pledget application. Note that sites 24 are in the area shown to be chemosensitive in adult cats (18, 20). Site 5 is at the level of the spinal cord. All fetuses used had undergone sinoaortic deafferentation. Cauterized areas are indicated by the shading.
heart rate, and respiration in response to the cyanide were recorded on a Beckman type R Dynograph recorder. Prior to every pledget application the control arterial pH and blood gas tensions were determined on a Radiometer microelectrode system at a temperature within 0.5”C of the lamb’s rectal temperature. A 15-min recovery period was observed between pledget applications to allow heart rate and blood pressure to return to base-line values. To hold diffusion artifacts to a minimum, the experiments were terminated when edema of the brainstem was noted by visual inspection. Control experiments either with soaked pledgets or flushing were also done with solutions of sodium chloride or glucose in mock CSF of similar tonicity to the cyanide solution (approx. 1,000 mosM). All solutions were prepared daily and the pH adjusted to 7.3-7.4 with HCl. In four fetuses the entire ventrolateral medullary surface on either side of the basilar artery was gently cauterized. After a 30-min recovery period, 2.5 mg of cyanide in 0.2 ml of solution was poured onto the surface of the medulla. The same dose was later injected into the femoral vein of three fetuses and in two fetuses the umbilical cord was clamped. At the end of the experiment the brain was removed and processed for microscopic estimation of the extent of the cautery. The response to cyanide shows no evidence of tachyphylaxis (4) and therefore pledgets were frequently applied several times to the same site during the experiment. When there was more than one application to a particular site the mean of any measured parameter (e.g., heart rate, blood gases, time to gasp) was taken as representative for the site. The cardiovascular changes were compared by the Student t-test of paired variates using only the first pledget application per site in each fetus. -
Only data from 12 of the 20 fetuses are included in this report. The other 8 fetuses either died during surgery or were discarded because of deteriorating blood gas tensions or evidence of brain edema. Of the 12 fetuses included, 8 were studied only before and 2 only after cautery of the medullary surface; 2 others were included in both groups. Histological examination of the cauterized brainstem revealed tissue destruction extending from 1 to 1.5 mm in depth over rnl:,st of the cauterized surface. Heart rate and blood pressure response ( Table 1). Application of cyanide-soaked pledgets to areas l-5 resulted in tachycardia and hypertension in a total of 84 of 93 trials (Figs. 2 and 3). The magnitude of this response was variable, 0 to >200 %, not only between different fetuses and sites but also between different applications to the same site. It is noteworthy that the heart rate and blood pressure responses were parallel and a stimulation of one and not the other was not observed. A depression of either parameter due to cyanide was also absent. Unilaterally applied cyanide produced a much reduced and inconsistent response. The presence or absence of respiratory efforts did not influence the effect on heart rate and blood pressure as indicated in Fig. 3. Although this response was elicited from all the sites tested, the greatest changes were observed when cyanide was applied to areas 2 and 3 nearest the roots of the cranial nerves IX to XI. Site 5 on the spinal cord was least responsive. The differences in response from sites 2 and 3 were statistically different from those obtained from site 5 (P < 0.01). I n addition, areas 2 and 3 also registered the shortest mean delay times to the onset and maximum of the response (Fig. 4). The mean (&SE) delay to the onset of blood pressure change from all areas, 14 & 1.60 s, was significantly shorter (P < 0.001) than that for the heart rate, 18 =t 1.75 s. Control trials with hypertonic glucose and NaCl solutions did not evoke any cardiovascular responses (Fig. 5). After cautery, cyanide still produced an elevation in both heart rate and blood pressure which in magnitude and response time was similar to that observed from area 5, on the cervical cord, which in these experiments was not cauterized. TABLE I. Blood pressure and heart rate response to topically applied cyanide to diJerent areas on surface of ventrolateral medulla and spinal cord SiteI% -I-
Arteria1
b1ood pres-
sure,
Heart min
mmHg
rate,
Values
beats/
expressed
1 2 3 4 5 1 2 3 4 5
Control I
6 9 9 9 8 6 9 9 9 8
1 I
C$($,
1 DifFerence
64zk16.99 76&14.6112&8.09 57~14.37 74h17.7417zk8.39 67zt20.22 88zk21.7821zk12.44 63zt14.29 73zt13.611Ozt10.23 64.zk15.61 7Oh17.24 6rt6.40 171*45.59222*44.2251*37.80 180rt40.32240~41.0960~29.71 184zt46.29249&44.5465rt42.63 1702t42.21211~46.0441~41.97 175zt31.09191rt23.6816~15.33
as means
rf= SD;
/
P
I -I-
PHYSIOLOGY
1975.
Prinledin
U.S.A.
Site of central chemosensitivity A. H. JANSEN
AND
Perinatal
Laboratory,
University
Research of
Manitoba,
V. CHERNICK Departments of Pediatrics and Physiology, Winnipeg, Manitoba R3E 0 WI, Canada
cannulated via a tracheostomy. Oxygen enriched air was supplied to keep the maternal arterial Po2 above 80 mmHg. Experiments were done on 20 fetal lambs ranging in weight from 2.9 to 5.9 kg. The preparation consisted of an exteriorized fetus with its placental circulation intact. Air inhalation was prevented by quickly capping the snout with a glove filled with warm water. The body temperature was held constant at 39-40°C with dry linen and a heating pad. A fetal femoral arterial catheter was connected to a pressure transducer (Statham P23Db) for continuous monitoring of the systemic blood pressure. The normal phasic blood pressure signal was electronically converted into a mean blood pressure tracing by an Electronics for Medicine recorder. Heart rate was measured by a cardiotachometer (Beckman 9857B) with needle electrodes placed under the skin on either side of the chest. The fetal trachea was cannulated without loss of tracheal fluid and connected to a liquid plethysmograph (14). This apparatus allowed the fetus to ventilate its lungs with liquid and in this respect simulate the intrauterine condition. The ventral surface of the brainstem was reached by ventral craniotomy (6) exposing the medulla oblongata from the pons to the spinal cord and laterally to the tympanic bullae. The dura was opened lengthwise and held back by wire springs. Thus the origins of the cranial nerve roots VI to XII were readily visible beneath the edge of the bone. Loss of CSF could not be prevented and therefore the brain surface was bathed in Mitchell’s mock CSF (18) prewarmed to body temperature. Blood loss from the surgical procedure was minimal. After the craniotomy, but just prior to opening the dura, peripheral chemo- and pressoreceptor function was eliminated in all fetuses studied by bilateral section of the sinus and vagus nerves. T o produce 1ocalized hypoxia, filter paper pledgets, 4 mm in diameter, were saturated in a solution of cyanide, 25 mg/ml of mock CSF, and initially placed unilaterally in a random sequence to discrete areas on the ventral surface of the medulla (Fig. 1). However since these trials were largely negative, in the experiments reported pledgets were applied bilaterally to corresponding sites. By calculation, the amount of cyanide held by two pledgets was approximately 0.45 mg, but since cyanide escapes readily into the air at the pH used, the actual dose reaching the brain surface was probably substantially less. After 2 min the pledgets were removed and the surface of the brain flushed with mock CSF. The mean arterial blood pressure output from the Electronics for Medicine recorder, the
JANSEN, A. H., AND V. CHERNICK. Site of central chemosensitiuity in fetal sheep. J. Appl. Physiol. 39( 1) : l-6. 1975.-The heart rate, blood pressure, and respiratory response to topically applied cyanide on the ventrolateral medullary surface and upper spinal cord was studied on exteriorized sinaortic-denervated fetal lambs under pentobarbital anesthesia. On all sites tested cyanide produced a rapid increase in heart rate and blood pressure (P < 0.05) which was most pronounced from the area adjacent to the nerve roots IX to XI (mean 32 %). Respiratory efforts consisting of l-8 gasps were induced in half the applications to the medulla but never when the pledgets were applied to the spinal cord. The mean delay to response was 43 s (range 13-l 02 s). After cautery of the chemosensitive areas, topical application of cyanide failed to stimulate gasping, whereas intravenous cyanide or cord clamping still produced a vigorous respiratory response. It is concluded that sympathetic stimulation of the heart and blood vessels can originate centrally in response to local histotoxic hypoxia of the ventral medulla and upper spinal cord. Furthermore, it is proposed that in the apneic fetus histotoxic hypoxia of the medulla initiates respiration possibly by stimulating a special gasping mechanism which is separate from the respiratory center responsible for rhythmic breathing after birth. The responsible neurons must be located at least 2 mm beneath the ventral medullary surface.
initiation medulla hypoxia; receptors
in fetal sheep
of fetal respiration; first breath; stimulation of ventral by cyanide; cardiovascular response to cyanide; fetal fetal central chemoreceptor; fetal peripheral chemo-
PREVIOUS EXPERIMENTS demonstrated that cyanide injected into the subarachnoid space of the ventral medulla of exteriorized fetal lambs will induce respiratory efforts and increase blood pressure and heart rate (16). This response was independent of the peripheral chemo- and pressoreceptors indicating a central effect of cyanide. However, the infusion technique used did not permit any conclusions regarding the exact site of action on the ventral surface of the brainstem nor whether the reactive cells were located near the surface or in the area of the respiratory center proper. The present study was undertaken to delineate the site of action of the cyanide more precisely. METHODS
Term pregnant ewes (Dorset-Suffolk) were with intravenous sodium pentobarbital, 15-20 wt, and maintained with additional injections, as required during the experiments. Catheters in a carotid artery and jugular vein and the
anesthetized mg/kg body 2-3 mg/kg, were placed trachea was 1
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
A. H. JANSEN
AND
V. CHERNICK
RESULTS
FIG. 1. Diagram of the ventral side of the medulla of the fetal sheep showing the roots of the cranial nerves V to XII and the areas of pledget application. Note that sites 24 are in the area shown to be chemosensitive in adult cats (18, 20). Site 5 is at the level of the spinal cord. All fetuses used had undergone sinoaortic deafferentation. Cauterized areas are indicated by the shading.
heart rate, and respiration in response to the cyanide were recorded on a Beckman type R Dynograph recorder. Prior to every pledget application the control arterial pH and blood gas tensions were determined on a Radiometer microelectrode system at a temperature within 0.5”C of the lamb’s rectal temperature. A 15-min recovery period was observed between pledget applications to allow heart rate and blood pressure to return to base-line values. To hold diffusion artifacts to a minimum, the experiments were terminated when edema of the brainstem was noted by visual inspection. Control experiments either with soaked pledgets or flushing were also done with solutions of sodium chloride or glucose in mock CSF of similar tonicity to the cyanide solution (approx. 1,000 mosM). All solutions were prepared daily and the pH adjusted to 7.3-7.4 with HCl. In four fetuses the entire ventrolateral medullary surface on either side of the basilar artery was gently cauterized. After a 30-min recovery period, 2.5 mg of cyanide in 0.2 ml of solution was poured onto the surface of the medulla. The same dose was later injected into the femoral vein of three fetuses and in two fetuses the umbilical cord was clamped. At the end of the experiment the brain was removed and processed for microscopic estimation of the extent of the cautery. The response to cyanide shows no evidence of tachyphylaxis (4) and therefore pledgets were frequently applied several times to the same site during the experiment. When there was more than one application to a particular site the mean of any measured parameter (e.g., heart rate, blood gases, time to gasp) was taken as representative for the site. The cardiovascular changes were compared by the Student t-test of paired variates using only the first pledget application per site in each fetus. -
Only data from 12 of the 20 fetuses are included in this report. The other 8 fetuses either died during surgery or were discarded because of deteriorating blood gas tensions or evidence of brain edema. Of the 12 fetuses included, 8 were studied only before and 2 only after cautery of the medullary surface; 2 others were included in both groups. Histological examination of the cauterized brainstem revealed tissue destruction extending from 1 to 1.5 mm in depth over rnl:,st of the cauterized surface. Heart rate and blood pressure response ( Table 1). Application of cyanide-soaked pledgets to areas l-5 resulted in tachycardia and hypertension in a total of 84 of 93 trials (Figs. 2 and 3). The magnitude of this response was variable, 0 to >200 %, not only between different fetuses and sites but also between different applications to the same site. It is noteworthy that the heart rate and blood pressure responses were parallel and a stimulation of one and not the other was not observed. A depression of either parameter due to cyanide was also absent. Unilaterally applied cyanide produced a much reduced and inconsistent response. The presence or absence of respiratory efforts did not influence the effect on heart rate and blood pressure as indicated in Fig. 3. Although this response was elicited from all the sites tested, the greatest changes were observed when cyanide was applied to areas 2 and 3 nearest the roots of the cranial nerves IX to XI. Site 5 on the spinal cord was least responsive. The differences in response from sites 2 and 3 were statistically different from those obtained from site 5 (P < 0.01). I n addition, areas 2 and 3 also registered the shortest mean delay times to the onset and maximum of the response (Fig. 4). The mean (&SE) delay to the onset of blood pressure change from all areas, 14 & 1.60 s, was significantly shorter (P < 0.001) than that for the heart rate, 18 =t 1.75 s. Control trials with hypertonic glucose and NaCl solutions did not evoke any cardiovascular responses (Fig. 5). After cautery, cyanide still produced an elevation in both heart rate and blood pressure which in magnitude and response time was similar to that observed from area 5, on the cervical cord, which in these experiments was not cauterized. TABLE I. Blood pressure and heart rate response to topically applied cyanide to diJerent areas on surface of ventrolateral medulla and spinal cord SiteI% -I-
Arteria1
b1ood pres-
sure,
Heart min
mmHg
rate,
Values
beats/
expressed
1 2 3 4 5 1 2 3 4 5
Control I
6 9 9 9 8 6 9 9 9 8
1 I
C$($,
1 DifFerence
64zk16.99 76&14.6112&8.09 57~14.37 74h17.7417zk8.39 67zt20.22 88zk21.7821zk12.44 63zt14.29 73zt13.611Ozt10.23 64.zk15.61 7Oh17.24 6rt6.40 171*45.59222*44.2251*37.80 180rt40.32240~41.0960~29.71 184zt46.29249&44.5465rt42.63 1702t42.21211~46.0441~41.97 175zt31.09191rt23.6816~15.33
as means
rf= SD;
/
P
I -I-
