Neutrophil mobility during anaesthesia in children. A trial of ascorbate premedication

In 20 healthy children undergoing elective surgery, mobility of neutrophils, both unstimulated and stimulated by endotoxin, was studied using a millipore filter system with microscopic determination of leading front migration. Paired samples were incubated with 10‐2 mol l‐1 calcium ascorbate and ten children also received 10 mg kg‐1 ascorbic acid before premedication. Stimulation of mobility was reduced after the opioid premedication (P < 0.05) in the ascorbate group only, hut not significantly during anaesthesia and surgery. A few individuals showed persisting abnormally low values. No effect of ascorbate in vivo or in vitro was demonstrated. There were no infrations.

Anaesthetic drugs, anaesthesia and surgery have been reported to reduce the mobility of neutrophils in adults (1-4) but not consistently so (5,6), and we know or no such studies in children. Surgical wound infection is associated with primary minor immunodeficiency (7), but it is not known whether slowing of neutrophils contributes too.
Ascorbic acid increases both random and directional migration of normal neutrophils in vztro, and both zn vztro and in uzvn the impaired mobility found in some children with recurrent bacterial infection (8-1 1 ) . We therefore studied neutrophil mobility in children undergoing anaesthesia and surgery, and the effects of zn uzvo and in vztro ascorbic acid.

PATIEN'I'S AND M E T H O D S
Twenty children wrre studied while undergoing general anaesthesia for a variety of elective procedures ('l'ahlr I ) . All werc free from cardiorespiratory, infrctivr or inflammatory illness and wcrc not receiving any medication 1)eforr the study. The parrnts gave informed consent and the study was approved by the hospital ethical rommittre. Patients were allocated randomly to receive either 10 mg kg-l ascorbic acid i.v. or the equivalent volume of saline, brfore premediration. Neither patient, parent, anaesthetists nor laboratory s t a r wrrr told tlir allocatioii.
On the morning of surgery an indwelling periphrral vcnous caw nula was placrd, through which first a blood sample was taken into preservative frer heparin, and then ascorbic acid or saline given.
Anaesthesia was induced with tliiopentonr 4-5 mg kg-' and maiutained with nitrous oxidr, oxygen and halothanr. Scventern children were intubated using suxamethonium 1-2 nig kg-' i.v. A third blood sample was taken about 5-10 min aftrr inductiorl but brfore surgical stimulation. Eight children thcn received tulxrurarinc 0.5 nig kg ~I and werr ventilated ('I'able I ) . A fourth sample was takrn at the cncl of surgrry before terminating anaesthesia or giving atropinr and neostigmine. 'Two cases (8 and 10, 'I'able I ) AS(J received local anaesthesia. No casr required blood transfusion during surgery. All were followed up for 7 days for evidence of infectiori. Nrutrophils for mobility measurement were srparated by drxtran sedimentation from 4-ml samples of Ileparinised blood. 'They w('re washed, thcn resuspended in Hank's solution at a coii(~riitratioii of 2 x lo6 m l -' and placrd abovr 3 pin milliporr membranes and iiicubated for 2 h. 'I'ht. migration of tlir leading fi.ortt of rieutropliils was measured microscopically as the mcan of fivr readings 011 each of duplicate membranes. l'hc chrmotactic f'actcir used was E. coli endotoxin-activated serum ( 1 2 ) . Neutrophil mobility was also mrasurrd with mol I -' calcium ascorbate in thr crll suspension. Control studies with cells from healthy adults wcrc' performed i n parallel.

Statistical methods
Results were evaluated with the paired arid unpaired Wilcoxori rdnksum tests, applying the Bonferroni corrections for mu1 tiple cornparisons. The level of significance chosen was P< 0.005.

RESULTS
Samples before premedication gave valurs or neutro-phi1 mobility comparable with our previous control series in adults and children. T h e concurrent series of 15 healthy adults who were not anaesthetised gave a meanks.d. or 110+ 14 pm for stimulated preparations, 52 & 15 pm when unstimulated, and 58 * 1 1 pm for net stimulation (endotoxin-stimulated minus unstimulated mobility). O u r two randomised groups did not differ significantly.
Stimulated mobility was reduced slightly in both groups after premedication from a mean of 103 to 97 pm in the ascorbate group and from 97 to 89 pm in the controls (n.s.) ( Table 2 ) . In two patients, stimulated mobility fell to abnormally low levels (52.2 and 56.3 The net stimulation values fell in both groups after premedication (Table 2), with a drop in mean value in the ascorbate group from 60.9 to 53.2 pm (P< 0.05).
During anaesthesia and surgery (Samples I11 and elm).
IV), group values were not significantly difrerent from their initial levels. Results varied widely ( Fig. 1 arid Stimulated mobility values which were clearly abnormal ( < 70 pm) were found in only three children after induction of anaesthesia, but in two these persisted throughout surgery. In vitro ascorbic acid did not produce a significant rise in mobility in any group. There were no difl'erences between the in uiuo ascorbate and placebo groups. No postoperative infections occurred.

2).
DISCUSSION O u r study demonstrated a signilicant depression of neutrophil mobility after premedication, an observation that does not seem to have been reported previously, but we railed to find the large reduction described in adults during anaesthesia (1). The effects seen during anaesthesia were less than we had expected from pilot studies in children, but in these we did not control for anaesthetic technique and used a shorter incubation.
Direct comparison of our data with thosr of other series is made difficult by the many differences in technique of' mobility measurement between reported studies. Aspects of our techniques may be sub-optimal for the detection of anaesthetic drug effects (4). I n particular, we made no attempt to preserve anaesthetic concentrations during incubation, but in the context of postoperative infection it was I'elt relevant to use a test capable of demonstrating efrects on mobility outlasting the exposure to anaesthetic agents. Anderson et al. (13) suggested that the various chemotactic agents may produce stimulation of mobility by direrent metabolic routes within the cell, so our results might have altered with an alternative chemoattractant. We did, however, demonstrate that the neutro- Table 2 Ncutrophil mobility (means and rangrs) of patients given (A) and not given (B) i.v. ascorbate before premedication. Samplcs takcn t)cli)rc ( I ) and after (11) premedication, after indurtion of anaesthesia (111) and at end of surgery (IV).
Neutrophil mobility (pm) Sample   phil mobility of some apparently normal children remained depressed during anaesthesia and surgery.
Although in vitro ascorbic acid stimulated neutrophils from patients with primary defects of mobility (8-lo), and there have been reports of this in healthy subjects (8, 1 l ) , we found no increase in our patients. Furthermore, in vivo pre-treatment with ascorbic acid did not appear to protect against this type of neutrophil depression. The only significant change found in our study was a reduction in mobility in the ascorbate group in Sample 11. Ascorbate has previously been reported to increase mobility in healthy subjects 1 h after intravenous injection (14), so it seems unlikely that the reduction we observed was due to the ascorbate.
In vitro exposure of neutrophils to anaesthetic agents can demonstrate pure drug effects, but the influence of in vivo administration occurs against a background Before premed After premed After induction End of surgery of the complex and variable endocrine stress response to anaesthesia and surgery. It is not known whether the hormonal changes which occur during anaesthesia in children (15) are associated with the neutrophil depression found in this study, but the need for a further investigation is suggested.