ACTA NEUROBIOL. EIXP. 1980, 40 : 985-992

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GENERATION OF RESPIRATORY PATTERN IN THE RABBIT -- BRAINSTEM TRANSECTIONS REVISITED

H. GROMYSZ and W. A. KARCZEWSKI

Department of Neurophysiology, Medical Research Center Polish Academy of Sciences, Warsaw, Poland

Abstract. The effects of unilateral transection of the lower brain- stem on the generation of central respiratory rhythm and its amplitude were studied in halothane anesthetized, vagotomized, paralyzed and artificially ventilated rabbits. Transections involving N. VII or rostra1 part of N. r VII elicited apneustic pattern of discharge in both phrenic nerves. Lesions made at more caudal levels (1.5-3.5 mm ros- tral to obex) restored a more normal pattern of discharge as far as frequency is concerned, but reduced the amplitude, particularly in the ipsilateral phrenic nerve. Sections below the obex abolished the activi- t y of the ipsilateral phrenic n. Since classical midpontine sections did not, elicit apneustic discharge, it is possible that this pattern is pro- duced by modifications in the number and functional connections of inspiratory-inhibitory neurons.

In a recent study (4) we have shown that focal, unilateral micro- injections of a local anesthetic (lignocaine) into brainstem structures (in which clusters of respiratory neurons could be demonstrated with microelectrodes) elicited reversible modifications in the pattern of fir- ing of both phrenic nerves. These effects were highly specific, i.e., the given pattern of discharge was produced exclusively from a strictly de- fined region of the lower brainstem but not from structures in which the respiratory units did not form clear-cut aggregates. Moreover, the type of disturbance depended strictly upon the type of activity (i.e., expiratory, inspiratory or mixed) found in the given region before the

blockade (4). Our results did not fully confirm the generally accepted concepts of the functional organisation of the brainstem respiratory network and we concluded that some regions or structures which were heretofore hardly regarded as important components of the respiratory network (e.g. Nucleus n. facialis) may be vital for the build-up of the proper respiratory pattern. We have therefore decided to confirm'or refute our conclusions by applying the classical technique of brainstem transections and comparing the effects with those elicited by focal microblockade.

The experiments were performed with 8 male rabbits weighing from 2.5 to 3.5 kg, vagotomized, anesthetized with neurolept analgesia (i.v. Fentanyl and Droperidol 0.1 and 0.5 mglkg, respectively) followed by halothane (0.7 vol percent in a 1 : 1 air-oxygen mixture), paralyzed with gallamine (5 mglkg) and artificially ventilated at eucarbic level (PaCoz 30-40 Torr). Arterial blood pressure, end-tidal COzQ/o and electri- cal activity of both phrenic nerves were continuously monitored and recorded at intervals from 5 to 10 min (Honeywell 4408A Visicorder, Tektronix 565 oscilloscope and ZRK RM-1040 magnetic recorder). Arterial blood samples were taken for pOz, pCOz and pH estimations (Radiometer BMS-1). When initial preparations were completed, the animal was reversed from supine to prone position and placed in a stereotaxic ap- paratus. An occipital craniotomy was made and the lower brainstem widely exposed. After recording the physiological parameters under control conditions transversal sections of the left half of the brainstem were performed with a 3 mm segment of a razor blade mounted in the holder of the stereotaxic frame's micromanipulator. The blade was lowered towards the base of the skull and the effects of a transection recorded immediately, and after 5, 10 and sometimes 20 min. The ce- rebellum was not removed. The transections were made, in varying sequences, at levels from 6 mm rostral to 1.5 mm caudal to obex. Experiments in which brain oedema, excessive bleeding or a rapid fall in blood pressure developed after an incision were discontinued. Each experiment was completed by fixing the brain in a 10°/o formaldehyde solution. After three days serial frozen sections (50 pm) were made and examined under a microscope to check the localisation, extent and completeness of the transections. The microscopic pictures were con- fronted with Messen and Olszewski's (8) and our own (Gromysz and Ruszczyk, unpublished) stereotaxic atlases.

Unilateral transections of the lower brainstem produced effects which (with one minor exception) correlated very well with the previously studied results of a focal lignocaine block (4). Transections at the level of Nucleus n. facialis (N. VII), i.e., 5.5 to 6.0 mm rostral to obex typical-

ly elicited apneustic discharges in both phrenic nerves (Fig. 1). This effect was only quantitatively enhanced on a few occasions when also the right half of the brainstem was separated from the more rostral structures. A similar, but less pronounced change was produced by

Fig. 1. Effects of a unilateral transection of the brainstem at the level of 5.5-6 mm rostral to obex (1 and 2 in Fig. 5). Traces from top to bottom: A "integrated" and B, directly recorded activity of the left phrenic nerve, C, directly recorded and D, "inte- grated" activity of the right phrenic nerve; E, refe- rence line of end-tidal COz (5O/o), F, arterial blood pressure; G, end-tidal COz. Horizontal bar, 1 s. I,

control; 11, 10 min after transection.

a transection 4 mm rostral to obex, i.e. between the caudal end of N. VII and rostral end of N. retrofacialis (N. rVII, Fig. 2). A qualitati- vely different picture followed transections at lower levels: when the caudal part of N. rVII or Nucleus ambiguus (A. c) was separated from more rostral structures the effects of such a transection consisted in an acceleration of the central respiratory rhythm with a concomitant decrease in the amplitude of inspiratory discharges forming thus a pat- tern resembling tachypnea (Fig. 3 11). When one of these lesions was

made on the background of already earlier elicited apneustic discharge, this abnormal pattern was immediately abolished. In contrast to the effect of lignocaine blockade, the transection affected more the ampli- tude of the ipsilateral phrenic nerve discharge. Transections below the obex (1.5 mm caudal) abolished the activity of the ipsilateral phrenic nerve (Fig. 3 111). Figure 4 presents the histologic cross sections through the lower brainstem (first section below the incision). The schematic representation of all levels of transection is shown in Fig. 5.

Fig. 2. Effect of a unilateral transection of the brainstem at the level 4 mm rostra1 to obex (3 in Fig. 5). Traces as in Fig. 1 except for E, 7.5O/0; F, end-tidal C02 and G, arterial blood pressure. I, control; 11, 10 min after transect-

ion.

All the main findings of the previous work in which aggregates of respiratory units were reversibly eliminated from the respiratory "controller" by lignocaine blocks (4) have been confirmed in the present study. Generally speaking, unilateral transections affecting N.VII (and

to some extent also the rostral part of N. rVII) elicited a considerable increase in the duration of inspiratory discharges in both phrenic ner- ves, just as was the case with the lignocaine block. Both groups of results strongly indicate that some inspiratory-inhibitory structure (or mecha- nism) is localized rostral to N. VII or, more precisely, to its lower half. This would obviously imply the rostral pons - Nucleus parabrachialis medialis (NPBM) and/or Kolliker-Fuse (2, 3). Surprisingly enough, ho- wever, when on a few occasions transections were performed just rostral

Fig. 3. Effects of unilateral transections of the brainstem at levels 1.5 mm rostral ( I i ) and 1.5 caudal (111) to obex. Traces as in Fig. 1 except for E, blood pressure and F, end-tidal CO,. I, control; 11, 10 min after first and 111, 10 min after second transect-

ion. See text.

to N. VII the pattern of discharge did not change in any appreciable way (Fig. 6). We are therefore inclined to think that the region of the lower brainstem between the sixth and ninth mm to obex is involved in switching the inspiration off in vagotomized rabbits even in the absence of hypercapnia believed by some authors (6, 14) to be prere- quisite for producing and maintaining apneustic breathing. The fact that a unilateral lesion can elicit apneustic respiration was shown both in animals (7) and in man ( lo) , despite the presence of intact vagi, but it seems to be disregarded by more recent authors. Obviously, since our rabbits were investigated during a relatively short time after the lesion, we do not know to what extent the apneustic type of inspirato- ry discharge would be reversible (as it frequently is - 13). Since, however, we have never observed any appreciable effects of halothane on breathing in rabbits, we can be at least convinced that the apneustic pattern was not a by-product of anesthesia (see 13, 15). There are several suggestions (with which we are inclined to agree) that apneusis is a functional disturbance, an epiphenomenon which should not be related to any anatomically defined "centers" (12). Although transec- tions separating the pons and the rostra1 medulla from the more caudal part of the respiratory network abolished in our experiments the apne- ustic, and restored the more eupnooic pattern of discharge in both phrenic nerves, we are not claiming a re-discovery of another "apneustic center" (caudal to the classical localisation). What we are suggesting is rather that lesion at this level affects the integrity and th-. functional configuration of the respiratory network, hypothetically by eliminating a certain number of inspiratory - inhibitory neurons and/or their specific connections. A similar view is expressed by St. John and Wang (14) in their study on decerebrate cats. These same authors have also found that more caudal lesions reduce the apneustic, and restore a more regular respiratory pattern. This was also a reproducible effect in our experiments when transectioiis were made caudally to N. rVII. Asym- metric effects elicited by such transections (e.g. 4 and 5 in Fig. 5) differ in one aspect from those obtained with the lignocaine block (4): the transection elicited larger decrease in ampli5.1de in the ipsilateral whereas blockade in the contralateral phrenic nerve. Both procedures did, however, similarly affect both the frequency and the pattern of discharge producing an equivalent of "rapid and shallow breathing". We suggest that some descending pathways are crossing at this level. A focal microblockade would made inactive first of all the crossing part of existing connections (and presumably a fraction of those uncro- ssing), whereas transection would eliminate all ipsilateral (uncrossing) connections, and, presumably, also some crossing fibres. This sugges-

Fig. 4. Eistolcgic cross section; through the lower brainstem. Czpitals A, B, C, etc. correspond to the levels of transection 1, 2, 3, etc. i n Fig. 5. Nurnbers denote neural structures (according to 8): 1, N. reticularis lateralis; 2, N. ret. parvocel:ularis; 3, N. ret. gig~ntccel lvlar is ; 4. N. n. hypoglossi; 5, N. tractus solitarii; 7, Genu n.; VII, N. n ; VII; 9, N. setrofacialis; 10, N. olivaris; 11, N. triangularis; 12, Subnucleus Deiters; 13, Subnucl. tractus spinalis n. trigemini; 14, Subnucleus reticularis ven-

tralis. First sfctior;.~ caudal to transections a r e presented.

Fig. 5. Schematic repre. sentations of transect- ions performed in the present study. Left half of the diagram shows for orientation some structures of the lower brainstem: N. V. mt. Nucleus motorius n. trigemini; N. VII, Nu- cleus n. facialis; N. rVII, Nucleus retrofa- cialis; A. c, Nucleus ambiguus. Horizontal lines, levels of transect- ions; vertical axis, mm caudal and rostral to

obex (V).

Fig. 6. Effect of a unilateral transection of the brainstem at the level just rostral to N.VII. Traces as in Fig. 2. 1;con- trol; 11, 10 min after transection. Note absence of appre- ciable changes in the pattern of respiratory discharge. See

text.

tion may be supported by our recent finding that a midsagittal section a t this level results in the appearance of two "independent" respiratory rhythms in both phrenic nerves, thus indicating the existence of an important connection of both halves of the brainstem at this level.

The disappearance of discharge in the ipsilateral phrenic nerve after the lowermost (caudal to obex) transection could be simply interpreted as the result of severing the descending axons from bulbo-spinal neu- rons to respiratory motoneurons. We have no data to support the view that these axons are crossing (9, 11) or to reject it (5). This old dis- crepancy (see also 1) will be perhaps clarified by a current study in which mid-sagittal incisions are combined with horizontal transections.

The authors a r e greatly indebted to Mrs. Krystyna Ruszczyk and Kinga Sro- czyriska for technical colla~boration and to Mrs. Barbara Sudziarska for typing the manuscript. This investigatian was supported by Project 10.4.2 of the Polish Academy of Sciences.

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Accepted 10 July 1980

H. GROMYSZ and W. A. KARCZEWL~KI, Medical Research Center, Polish Academy of Sc~ences , Dworkowa 3, 00-784 !Warsaw, Poland.