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Neonatal animals such as the kitten and the rodent have proven to be useful models to study the effect of sensory inputs on CNS development. These models have clearly indicated that sensory signals are required during critical developmental periods for proper CNS maturation(1–8). In humans, clinical studies have demonstrated various effects of tactile stimulation during infancy(9–12). Field et al.(10), for example, have shown that tactile stimulation in hospitalized preterm infants results in greater weight gain and higher Brazelton behavioral scores.

Recently, we have studied the role of EGF as a regulator of energy metabolism in the newborn period. We found that EGF administration to newborn rats increased serum lactate concentrations, decreased systemic oxygen consumption, and had little effect on acid-base balance(13). The effect on serum lactate concentration was particularly intriguing insofar as several laboratories have reported that lactate is an important metabolic fuel for the developing brain in the newborn rat. Given the choice among lactate, glucose, or ketone bodies, as metabolic substrates, the neonatal rat brain prefers lactate(14–18).

Administration of EGF also had a notable effect to lower body temperature(19). Rapid reduction in body and brain temperature have been reported after TS of newborn rats by gentle stroking(20–22). Given the dual effect of EGF to lower body temperature and to raise serum lactate concentration, we tested the hypothesis that TS would increase lactate concentrations in the newborn rat.

METHODS

Animals. Pregnant Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienople Park, PA) were obtained on the 15th d of gestation and housed singly in our animal facility. Normal light:dark cycles (12 h:12 h) were maintained, and rat chow and water were provided ad libitum. After birth, litters were divided according to the experimental design into appropriate treatment groups. When required, individual pups were distinguished by coded, s.c. tattoos with India ink over the low dorsal region. Animals were classified as d 0, 1, 2, etc., based on 24-h postnatal intervals. Pups were removed from the nest preceding each experiment and were weighed on a top-loading balance (Mettler, model PE-360). Individual animals were placed in perforated Styrofoam cups in an infant incubator (Air-Shields, model C100) controlled at nest temperature (34 °C)(13). Selected experiments were performed at room temperature (22 °C). All studies were approved by the Institutional Animal Care and Use Committee.

TS regimen. After a 60-min adaptation period, a standard regimen of TS was applied in all experiments(23). The regimen consisted of holding all pups gently between the thumb and forefinger of one hand. Pups receiving TS were stroked on the dorsal surface in a rostral to caudal direction with a soft camel hair brush. Pups were stroked with a rate of one stroke/s for 30 s followed by a 30-s rest period every minute for a total of 10 min. This procedure was used to mimic nonspecific maternal stimulation, such as licking and handling of the pups(20, 23). After TS, pups were sacrificed by decapitation at various time intervals as determined by the experimental design.

Laboratory measurements. Arteriovenous blood samples were collected in microcentrifuge tubes and centrifuged at 4 °C. Ten microliters of the serum were used for the measurement of glucose and lactate using a glucose-lactate analyzer (Yellow Springs Instruments, model 2300 STAT). In separate experiments, mixed arteriovenous blood was collected using a capillary kit (Ciba-Corning, model 4718190) and blood gases were measured on a blood gas analyzer (Ciba-Corning, model 178).

Pharmacologic studies. EGF. Treated animals received 500 ng/g BW of receptor-grade EGF (BioProducts for Science, Indianapolis, IN) as a daily s.c. injection over the flank or low dorsal region using a 10-mL Hamilton syringe as described previously(19). Control animals received an equivalent volume of saline. EGF injections were administered within 12 h of birth (d 0) and repeated every 24 h for a total of three doses. Both control and EGF pretreated animals were further subdivided into two groups; one received TS and the other did not. Serum lactate and glucose determinations were obtained on the 3rd or 4th d of life immediately after the TS regimen.

Pentobarbital. Study pups (28-40 h of age) were anesthetized with 15 mg/g BW pentobarbital (Abbott Laboratories, North Chicago, IL) via s.c. injection. Control pups received an equivalent volume of saline. The standard TS regimen was performed on both control and treated pups, approximately 20 min after onset of anesthesia. Immediately after TS, serum was collected for determination of glucose and lactate concentrations.

Capsaicin. Capsaicin (Sigma Chemical Co., St. Louis, MO) was dissolved in a vehicle of 10% ethanol and 10% Tween 80 in normal saline. Between 26 and 40 h of age, pups received a single, s.c. injection with 50 mg/g BW. Control animals received an equivalent volume of vehicle. On d 3 or 4 of life, tactile stimulation was administered using the standard regimen. Pups were sacrificed, and serum was obtained for the determination of lactate and glucose concentrations.

Statistical analysis. Data were analyzed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). All analyses were performed using unpaired two-tailed t tests with a significance level set at p < 0.05. If variances were unequal or the data were not normally distributed, the Mann-Whitney rank sum test was used to test for statistical significance. All results are expressed as mean ± SEM.

RESULTS

Figure 1 shows the normal ontogeny of serum lactate level in millimoles per liter after birth in the rat. Levels are high immediately after birth and undergo a serial fall over the 1st wk of life. TS, therefore, acts upon a dynamic background of progressively falling serum lactates. As shown in Figure 2, serum lactate levels in newborn (d 0) rats were significantly higher in the stimulated group compared with controls (5.4 ± 0.3 mM versus 2.6 ± 0.2 mM, respectively, mean ± SEM, p < 0.01, n = 37). By 1 wk of life (d 7), TS had no significant effect on serum lactate (1.6± 0.1 mM versus 1.4 ± 0.1 mM, p = 0.20,n = 16). Figure 3 reveals the effect of TS on serum lactate at nest and room temperatures. Pups maintained at either 34 or 22 °C showed a significant rise in lactate after TS. Equilibration at 22°C resulted in lower lactate levels for both controls and TS pups relative to nest temperature. Control values at 34 and 22 °C were 2.5 ± 0.9 mM and 1.8 ± 0.1 mM, respectively. TS values for the same temperatures were 7.0 ± 0.4 mM and 5.0 ± 0.5 mM, respectively.Figure 4 depicts experiments in which tactile stimulation was performed per protocol on 38 newborn rat pups. Blood was collected at 0, 10, or 30 min after stroking. Serum lactate levels remained significantly elevated for 30 min after cessation of stimulation. Serum lactate levels at 0, 10, and 30 min were 4.7 ± 0.3 mM, 4.0 ± 0.2 mM, and 3.4 ± 0.2 mM, respectively, for stimulated pups, whereas the mean baseline level for controls at all time points was 2.5 ± 0.1 mM, p < 0.01.

Figure 1
figure 1

Ontogeny of serum lactate over the 1st wk of life in the rat. Lactate levels are high immediately after birth and decrease rapidly over the first few hours. The rate of decrease diminishes as the animal matures. Values are expressed as means ± SEM with the number of animals shown in parentheses.

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Figure 2
figure 2

Effect of TS on lactate levels in newborn (d 0) and 1-wk-old rat pups. TS significantly elevated serum lactate levels in the newborn animals but not in the older group; *p < 0.01.

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Figure 3
figure 3

Effect of TS on serum lactate at different ambient temperatures. Equilibration at 22 or 34 °C for 1 h resulted in elevation in lactate after TS; *p < 0.01. Both baseline and poststimulation values were decreased at the lower ambient temperature.

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Figure 4
figure 4

Duration of serum lactate elevation after TS. Serum lactate levels were determined 0, 10, and 30 min after cessation of TS. Lactate levels remained significantly elevated above baseline values at all points; *p < 0.01.

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In other experiments, mixed arteriovenous blood samples were collected, and blood gas data were compared. Pups were allowed to recover for 5 min after the cessation of TS before sacrifice. Table 1 shows the results obtained from 42 newborn pups. TS resulted in a significant increase in pH and oxygen saturation, and a decrease in Pco2 and base excess.

Table 1 Effect of TS on mixed arteriovenous blood gases
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Experiments were also performed to evaluate the effects of selected pharmacologic agents on circulating lactate and the TS response. In earlier work, we demonstrated that systemic administration of EGF acutely (within 4 h) increased serum lactate levels in neonatal rats(13). In the present study, chronic EGF pretreatment resulted in a significant increase in serum lactate concentrations in nonstimulated EGF-pretreated newborn rats when compared with nonstimulated saline-pretreated animals (2.5 ± 0.1 mM versus 2.0 ± 0.1 mM, respectively, p < 0.05)(Table 2a). Tactile stimulation of EGF-pretreated animals resulted in an augmented but not statistically significant rise in serum lactate concentrations when compared with stimulated saline-treated animals(6.4 ± 0.4 mM versus 5.7 ± 0.6 mM, respectively)(Table 2a).

Table 2 Effects of pharmacologic agents on serum lactate(mM) (mean ± SEM)
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Pentobarbital anesthesia was administered to evaluate a potential role for the CNS in mediating the elevated lactate response to tactile stimulation. Pretreatment with pentobarbital markedly diminished the effect of TS on serum lactate. Additional experiments were performed to test whether type C unmyelinated nerve fibers are required to mediate the lactate response to TS. For this purpose, newborn pups were treated with the neuromodulator, capsaicin, which results in rapid and irreversible destruction of these nerve fibers in neonatal but not adult animals(24). Pretreatment with capsaicin did not block serum lactate elevation after TS (Table 2c).

Glucose levels were assayed concurrently with lactate in all experiments. Serum levels of glucose exhibited a general ontogenetic increase from 4.7± 0.1 mM (n = 23) on the first postnatal day to 7.2 ± 0.2 mM (n = 13) on d 7 of life. This finding is consistent with previous reports of increasing glucose levels with advancing age(25, 26). No significant differences in serum glucose were observed after TS.

DISCUSSION

There is growing evidence demonstrating that extremely specific sensory cues regulate different physiologic and behavioral responses in young animals. Studies have shown, for example, that the development of the immature CNS is critically dependent upon sensory input during the immediate postnatal period. Experimental interference with several sensory modalities such as vision, touch, and hearing result in profound anatomical, functional, and biochemical impairment of the CNS structures that regulate such modalities(5–8).

In newborn rats, TS is an important regulator of somatic growth. Studies by Schanberg et al.(23) and Butler et al.(27) have clearly linked TS with the regulation of ODC in neonatal rodents. ODC is a sensitive index of maturation and growth of internal organs such as the heart, liver, and brain. Brain, liver, and heart ODC activity are decreased by 35, 81, and 53%, respectively, when rat pups were removed from their mother for periods as short as 1 h. ODC activity normalizes quickly after pups are returned to the mother or provided with nonspecific tactile stimulation(23, 27).

Our findings extend the Schanberg model and demonstrate that TS elevates serum lactate concentrations. These results raise the question of what mechanisms regulate serum lactate in the newborn period. Unlike glucose, which is under tight hormonal control, the regulation of serum lactate is less clear. In the fetus, lactate is produced by the placenta(28). Fetal rat brain slices show a high capacity for lactate oxidation in vitro during late gestation(18). This capacity remains high during the early postnatal period(17). The brain of the early suckling rat, for example, utilizes lactate in preference to other fuels such as glucose and 3-hydroxybutyrate(15, 16). These findings suggest that lactate may play a vital role as an energy substrate in the brain during the early neonatal period.

Serum lactate levels, like other metabolic fuels, are a dynamic function of the rate of production and the rate of consumption. The production of lactate by muscle during anaerobic conditions, for example, has been well studied(29). Our results, theoretically, may be explained on the basis of a sensorimotor reflex arc in which TS activates muscular movement with consequent lactate generation and release. This interpretation is consistent with a mechanism by which environmental cues, such as touch, can regulate muscle activity, lactate generation, and consequent levels of circulating lactate.

At least six entirely different types of tactile receptors are known, with the cruder types of tactile signals being transmitted via the slower unmyelinated type C nerve fibers(30). Pretreatment with capsaicin did not block lactate elevation after TS (Table 2c). This suggests that type C fibers are not involved in the mediation of the elevated lactate response, but does not exclude mediation by other nerve fibers types. Pretreatment with pentobarbital markedly diminished the effect of TS on serum lactate, supporting a role for CNS mediation (Table 2b).

Alternate mechanisms contributing to the control of circulating lactate can also be hypothesized. Parturition marks a major transition in resting metabolism. In both humans and rats, circulating lactate levels undergo a slow but progressive postnatal fall(16, 31). High lactate levels near the time of birth have been speculated to result from fetal hypoxia with a resulting metabolic lactic acidosis. In this study, blood gas data (Table 1) show mild, but significant, increases in pH and in base deficit and a decrease in the Pco2 with no evidence of hypoxia. These findings may indicate a stimulation in respiratory effort secondary to tactile stimulation accompanied by a significant rise in circulating lactate levels, possibly due to skeletal muscle activation. Lower ambient temperatures reduced baseline levels of lactate in control pups but did not abrogate the response to TS (Fig. 3). These findings are consistent with a general decrease in motor responsiveness.

In earlier work, we demonstrated an increase in serum lactate concentrations under aerobic conditions in the newborn rat by pharmacologic treatment with EGF(13). This finding supports a role for mammalian growth factors like EGF in metabolic control. Other possible mechanisms for lactate generation include local production from extramuscular sources such as the liver or the skin. Many studies have demonstrated, for example, that cutaneous breakdown of glucose proceeds to lactate under aerobic conditions despite the presence all the enzymatic machinery for oxidation via the tricarboxylic acid cycle(32, 33). In vitro culture of skin biopsies from EGF-treated rat pups results in elevation in media lactate. Similar culture of skin biopsies from pups receiving TS, however, showed no such elevation (data not shown).

In this study, we were guided in our initial experimental design by the possibility that lactate elevations were important for perinatal brain metabolism in the rat. The importance of lactate to the newborn rat brain during the critical early neonatal period is well established. Elevated serum lactate, however, does not mean an increase in the consumption or utilization of lactate by the brain. Proof of lactate uptake by the CNS requires demonstration of cross-brain arteriovenous differences. Nevertheless, our results clearly indicate that TS during a critical period of development results in sustained elevation in circulating lactate levels in the newborn rat. We speculate that the transduction of sensory signals by the skin in the newborn rat (from mechanical contact with the mother or other littermates) is an important mechanism for supplying the developing brain with its preferred metabolic substrate, i.e. lactate.