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Neonatal halothane anesthesia affects cortical morphology
Developmental Brain Research 124 (2000) 121–124 www.elsevier.com / locate / bres
Short communication
Neonatal halothane anesthesia affects cortical morphology ˜ Joseph L. Nunez, Janice M. Juraska* Neuroscience Program and Department of Psychology, University of Illinois at Champaign-Urbana, 603 E. Daniel Street, Champaign, IL 61820, USA Accepted 15 August 2000
Abstract Neonatal cryoanesthesia has recently been documented to affect morphology and behavior after a single exposure [Dev. Brain Res. 111 (1998) 89; Horm. Behav. 37 (2000) 169]. In the current experiment, we investigated the effect of one-time exposure to halothane inhalant anesthesia on neonatal rats of both sexes. Fifteen minutes of exposure on postnatal day one resulted in detectable changes in the volume of the visual cortex at 3 months. Thus, neonatal halothane alters neural development and its effects are observable in the adult rat. 2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cerebral cortex and limbic system Keywords: Development; Rat; Surgery
Surgical procedures such as gonadectomies are often performed on neonatal rats. These operations require the use of anesthetics; hypothermia [8,10,13] or inhalants (e.g., halothane and isoflurane) [1,7,13] are often used. While these anesthetics have been demonstrated to be effective on neonatal pups [13], recent work from our lab has shown that neonatal cryoanesthesia permanently alters the cerebral cortex and hippocampus [4,5]. We found that 60 min of cryoanesthesia (4–58C) on postnatal day 1 resulted in a 13–23% (depending upon the sex of the animal) loss of cells in layer 2 / 3 of the primary visual cortex relative to control animals [4]. An 8–10% decrease in the total volume of the hippocampus [5] was also found, along with significant decrements in water maze performance [5]. As little as 15 min of cold exposure decreased the volume of the visual cortex by 5–10% [4]. Following neonatal cryoanesthesia, the sizable (19%) sex differences in visual cortex neuron number and volume [9] were diminished or abolished. However, it is unclear whether these effects are specific to cryoanesthesia or apply to other general anesthetics. Previous work has demonstrated that halothane inhalant *Corresponding author. Tel.: 11-217-333-8546; fax: 11-217-2445876. E-mail address: [email protected] (J.M. Juraska).
exposure, when administered chronically at subanesthetic levels to developing rats, causes decreases in synaptic density [12] and the size of the dendritic tree [11] in the cerebral cortex. Likewise, decrements in the dendritic tree of rat hippocampal neurons have been observed following short-term exposure to pentobarbital [3]. However, this work does not preclude the use of similar anesthetics for one-time neonatal surgical operations of short duration. In the present study, we investigated the possible effects of using halothane compared to a local anesthetic for neonatal surgical procedures. Rats of both sexes were administered halothane or a subcutaneous injection of lidocaine on postnatal day 1, in a manner equivalent to that used for surgical manipulations [5,7], and the volume of the primary visual cortex was assessed in adulthood. Long-Evans hooded rats (obtained from Simonsen Lab, Gilroy, CA) were mated in the Psychology Department breeding colony. The day following the appearance of the sperm plug was designated as gestational day 1. All animals were housed under a 12:12-h light / dark cycle, with food and water provided ad libitum. The experimental (halothane exposed) and lidocaine (local anesthetic) animals came from different litters. All animals were time bred and a subset of each litter was removed from their mothers at 1.5 h after birth. The experimental males and females (seven littermate sets)
0165-3806 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00093-6
122
˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
were exposed to halothane through a surgical aspirator with a nozzle end. At an oxygen flow rate of 1 l / min, 2% halothane was administered to the pups for 3 min. The halothane concentration was then reduced to 1% for 12 min. This procedure mimics the anesthesia required for neonatal gonadectomies. After 10 min of exposure, the rats were given a subcutaneous injection of sesame oil (0.1 ml). The lidocaine males and females (eight littermate sets) were subcutaneously implanted with a cholesterol pellet using a local injection (0.01 ml) of lidocaine (20 mg / ml) as anesthetic. These animals also served as controls in another study of hormone manipulations [6]. All procedures were performed on a heated pad (35–378C), followed by housing in an incubator (35–408C) prior to return to their mothers. Pups from both groups were weaned on day 25 and housed with one or two same-sex littermates until sacrifice at 85–90 days of age. Body weights were taken prior to sacrifice. Under deep anesthesia, the rats were perfused intracardially with room temperature Ringer’s solution, followed by 4% paraformaldehyde in 0.2 M phosphate buffer solution and the brains were removed. The brains were post-fixed for 7 days and coded to avoid experimenter bias. Brain weights were taken at this time. The brains were sectioned with a tissue chopper into coronal blocks containing the visual cortex, stored in 30% sucrose in fixative for 3 days and then sectioned coronally on a freezing microtome at 60 mm. Sections were mounted immediately after slicing, then dried and stained with methylene blue–azure II for cytoarchitectonic analysis. Drawings of the monocular (Oc1M) and binocular (Oc1B) subfields of the rat primary visual cortex were made from each animal from four tissue sections spaced at regular anterior to posterior intervals using a camera lucida at 360 magnification. For the planes drawn and details of ˜ et al. parcellation of the primary visual cortex, see Nunez [4]. In order to obtain the volume of each subfield (Oc1M and Oc1B), each of the four planes was digitized into a computer to find its area. This area was then multiplied by the thickness of the sections following it to the next measured plane to obtain a regional volume. All four regional volumes were added to find the total volume of Oc1M and Oc1B. Two way analyses of variance (group, sex) were performed on measures of brain and body weight. Because the halothane and lidocaine animals came from different litters, planned comparisons [2] within each group were run on measures of visual cortex (Oc1M and Oc1B) volume. There was a near-significant effect of group on adult brain weight (P50.059), with lidocaine-injected animals having heavier brain weights than experimental animals (Fig. 1A). There was also a significant main effect of sex on adult brain weight (P50.0001) in both groups. There was a significant main effect of group on adult body weight (P50.033), with halothane exposed animals
Fig. 1. Effect of halothane versus lidocaine exposure upon brain (A) and body (B) weight in the adult rat. There was a significant main effect of group on body weight (P50.033), and a trend on brain weight (P5 0.059). There was a significant main effect of sex on both brain and body weight (P50.0001 for each).
having heavier body weights than lidocaine injected animals (Fig. 1B). There was a significant main effect of sex on adult body weight (P50.0001) in both groups. For Oc1M volume, there was a significant effect of sex in the lidocaine-injected animals (P50.0001), with males having a larger Oc1M volume than females, but not in the halothane-exposed animals (P50.819) (Fig. 2A). For the volume of Oc1B, lidocaine injected males had a significantly larger volume than the lidocaine injected females (P50.0001). This was not found between the halothane exposed males and females (P50.986) (Fig. 2B). The present experiment demonstrated that halothane, a commonly used anesthetic for neonatal procedures such as gonadectomies, alters cortical morphology in rats and abolishes sex differences after a single exposure. In the
˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
123
the effects were not mediated by general metabolism or overall growth. Also, large sex differences were found in these measures in the halothane exposed rats compared to the lidocaine exposed animals. The visual cortical volumes show an entirely different pattern. The sizable sex difference seen in lidocaine exposed rats was not observed in the halothane exposed rats. The relatively minor effects on brain weight and the persistence of sex difference in brain weight indicates that halothane effects, like cryoanesthesia, are regionally specific [1,5]. It is interesting that the cerebral cortex is affected by both anesthetics. Halothane appears to have a differential effect upon the sexes, in spite of equivalent exposure time. While neonatal halothane exposure in males decreased visual cortex volume by 6–12% relative to lidocaine-injected males, halothane exposure in females increased volume 11–17% relative to lidocaine-exposed females. This study highlights the deleterious effects of halothane, a commonly used anesthetic, upon cortical morphology in neonates. A single 15-min exposure to halothane on day 1 eliminated the sex difference observed in the adult rat primary visual cortex. These data indicate that the use of any anesthetic in the neonatal rat may alter neural morphology.
Acknowledgements This work was supported by NSF IBN 9723918 to J.M.J. J.L.N. was supported by a predoctoral fellowship NIGMS 1 F31 GM17209.
References
Fig. 2. Effect of halothane versus lidocaine exposure upon the total volume of Oc1M (A) and Oc1B (B) in the adult rat. There was a significant effect of sex for both Oc1M and Oc1B in the lidocaine injected animals (P50.0001 for both measures), but not in the halothane exposed animals.
absence of any anesthesia in a prior study [9] and in the present study when neonatal rats were given only to a local injection of lidocaine, sex differences were found in the volume of the monocular (Oc1M) and binocular (Oc1B) subfields of the rat primary visual cortex. It does not appear that neonatal lidocaine injections influenced the sex difference in visual cortex volume because the present study documented a 22% difference between males and females in Oc1B, and a 19% difference was previously found in Oc1B in our lab [9]. Neonatal halothane does not appear to affect all measures in the same way or degree. Body weight was minorly (but significantly) higher, while brain weight was somewhat smaller in halothane exposed rats. This indicates that
[1] M.F. Eckenhoff, R.G. Eckenhoff, Quantitative autoradiography of halothane binding in rat brain, J. Pharmacol. Exp. Ther. 285 (1998) 371–376. [2] W.L. Hayes, Comparisons among means, in: W.L. Hayes (Ed.), Statistics, Harcourt Brace College, Fort Worth, 1994, pp. 423–471. [3] C.D. Jacobson, L.L. Antolick, R. Scholey, E. Uemura, The influence of prenatal pentobarbital exposure on the growth of dendrites in the rat hippocampus, Dev. Brain Res. 44 (1988) 233–239. ˜ [4] J.L. Nunez, B.Y. Kim, J.M. Juraska, Neonatal cryoanesthesia. affects the morphology of the visual cortex in the adult rat, Dev. Brain Res. 111 (1998) 89–99. ˜ [5] J.L. Nunez, W.A. Koss, J.M. Juraska, Hippocampal anatomy and water maze performance are affected by neonatal cyroanesthesia in rats of both sexes, Horm. Behav. 37 (2000) 169–178. ˜ [6] J.L. Nunez, J. Sodhi, J.M. Juraska, Neonatal androgens affect neuron number in the adult male and female rat visual cortex. Cereb. Cortex (in press, pending revision). [7] C.M. Park, K.E. Clegg, C.J. Harvey-Clark, M.J. Hollenberg, Improved techniques for successful neonatal rat surgery, Lab. Anim. Sci. 42 (1992) 508–513. [8] C.B. Phifer, L.M. Terry, Use of hypothermia for general anesthesia in preweanling rodents, Physiol. Behav. 38 (1986) 887–890. [9] S.N.M. Reid, J.M. Juraska, Sex differences in neuron number in the binocular area of the rat visual cortex, J. Comp. Neurol. 321 (1992) 448–455.
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˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
[10] H.L. Rosomoff, D.A. Holaday, Cerebral blood flow and cerebral oxygen consumption during hypothermia, Am. J Physiol. 179 (1954) 85–88. [11] E. Uemura, W.P. Ireland, E.D. Levin, R.E. Bowman, Effects of halothane on the development of rat brain: a golgi study of dendritic growth, Exp. Neurol. 89 (1985) 503–519.
[12] E. Uemura, R.E. Bowman, Effects of halothane on cerebral synaptic density, Exp. Neurol. 69 (1980) 135–142. [13] D.K. Yi, G.A. Barr, The suppression of formalin-induced c-fos expression by different anesthetic agents in the infant rat, Dev. Psychobiol. 29 (1996) 497–506.
Short communication
Neonatal halothane anesthesia affects cortical morphology ˜ Joseph L. Nunez, Janice M. Juraska* Neuroscience Program and Department of Psychology, University of Illinois at Champaign-Urbana, 603 E. Daniel Street, Champaign, IL 61820, USA Accepted 15 August 2000
Abstract Neonatal cryoanesthesia has recently been documented to affect morphology and behavior after a single exposure [Dev. Brain Res. 111 (1998) 89; Horm. Behav. 37 (2000) 169]. In the current experiment, we investigated the effect of one-time exposure to halothane inhalant anesthesia on neonatal rats of both sexes. Fifteen minutes of exposure on postnatal day one resulted in detectable changes in the volume of the visual cortex at 3 months. Thus, neonatal halothane alters neural development and its effects are observable in the adult rat. 2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cerebral cortex and limbic system Keywords: Development; Rat; Surgery
Surgical procedures such as gonadectomies are often performed on neonatal rats. These operations require the use of anesthetics; hypothermia [8,10,13] or inhalants (e.g., halothane and isoflurane) [1,7,13] are often used. While these anesthetics have been demonstrated to be effective on neonatal pups [13], recent work from our lab has shown that neonatal cryoanesthesia permanently alters the cerebral cortex and hippocampus [4,5]. We found that 60 min of cryoanesthesia (4–58C) on postnatal day 1 resulted in a 13–23% (depending upon the sex of the animal) loss of cells in layer 2 / 3 of the primary visual cortex relative to control animals [4]. An 8–10% decrease in the total volume of the hippocampus [5] was also found, along with significant decrements in water maze performance [5]. As little as 15 min of cold exposure decreased the volume of the visual cortex by 5–10% [4]. Following neonatal cryoanesthesia, the sizable (19%) sex differences in visual cortex neuron number and volume [9] were diminished or abolished. However, it is unclear whether these effects are specific to cryoanesthesia or apply to other general anesthetics. Previous work has demonstrated that halothane inhalant *Corresponding author. Tel.: 11-217-333-8546; fax: 11-217-2445876. E-mail address: [email protected] (J.M. Juraska).
exposure, when administered chronically at subanesthetic levels to developing rats, causes decreases in synaptic density [12] and the size of the dendritic tree [11] in the cerebral cortex. Likewise, decrements in the dendritic tree of rat hippocampal neurons have been observed following short-term exposure to pentobarbital [3]. However, this work does not preclude the use of similar anesthetics for one-time neonatal surgical operations of short duration. In the present study, we investigated the possible effects of using halothane compared to a local anesthetic for neonatal surgical procedures. Rats of both sexes were administered halothane or a subcutaneous injection of lidocaine on postnatal day 1, in a manner equivalent to that used for surgical manipulations [5,7], and the volume of the primary visual cortex was assessed in adulthood. Long-Evans hooded rats (obtained from Simonsen Lab, Gilroy, CA) were mated in the Psychology Department breeding colony. The day following the appearance of the sperm plug was designated as gestational day 1. All animals were housed under a 12:12-h light / dark cycle, with food and water provided ad libitum. The experimental (halothane exposed) and lidocaine (local anesthetic) animals came from different litters. All animals were time bred and a subset of each litter was removed from their mothers at 1.5 h after birth. The experimental males and females (seven littermate sets)
0165-3806 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00093-6
122
˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
were exposed to halothane through a surgical aspirator with a nozzle end. At an oxygen flow rate of 1 l / min, 2% halothane was administered to the pups for 3 min. The halothane concentration was then reduced to 1% for 12 min. This procedure mimics the anesthesia required for neonatal gonadectomies. After 10 min of exposure, the rats were given a subcutaneous injection of sesame oil (0.1 ml). The lidocaine males and females (eight littermate sets) were subcutaneously implanted with a cholesterol pellet using a local injection (0.01 ml) of lidocaine (20 mg / ml) as anesthetic. These animals also served as controls in another study of hormone manipulations [6]. All procedures were performed on a heated pad (35–378C), followed by housing in an incubator (35–408C) prior to return to their mothers. Pups from both groups were weaned on day 25 and housed with one or two same-sex littermates until sacrifice at 85–90 days of age. Body weights were taken prior to sacrifice. Under deep anesthesia, the rats were perfused intracardially with room temperature Ringer’s solution, followed by 4% paraformaldehyde in 0.2 M phosphate buffer solution and the brains were removed. The brains were post-fixed for 7 days and coded to avoid experimenter bias. Brain weights were taken at this time. The brains were sectioned with a tissue chopper into coronal blocks containing the visual cortex, stored in 30% sucrose in fixative for 3 days and then sectioned coronally on a freezing microtome at 60 mm. Sections were mounted immediately after slicing, then dried and stained with methylene blue–azure II for cytoarchitectonic analysis. Drawings of the monocular (Oc1M) and binocular (Oc1B) subfields of the rat primary visual cortex were made from each animal from four tissue sections spaced at regular anterior to posterior intervals using a camera lucida at 360 magnification. For the planes drawn and details of ˜ et al. parcellation of the primary visual cortex, see Nunez [4]. In order to obtain the volume of each subfield (Oc1M and Oc1B), each of the four planes was digitized into a computer to find its area. This area was then multiplied by the thickness of the sections following it to the next measured plane to obtain a regional volume. All four regional volumes were added to find the total volume of Oc1M and Oc1B. Two way analyses of variance (group, sex) were performed on measures of brain and body weight. Because the halothane and lidocaine animals came from different litters, planned comparisons [2] within each group were run on measures of visual cortex (Oc1M and Oc1B) volume. There was a near-significant effect of group on adult brain weight (P50.059), with lidocaine-injected animals having heavier brain weights than experimental animals (Fig. 1A). There was also a significant main effect of sex on adult brain weight (P50.0001) in both groups. There was a significant main effect of group on adult body weight (P50.033), with halothane exposed animals
Fig. 1. Effect of halothane versus lidocaine exposure upon brain (A) and body (B) weight in the adult rat. There was a significant main effect of group on body weight (P50.033), and a trend on brain weight (P5 0.059). There was a significant main effect of sex on both brain and body weight (P50.0001 for each).
having heavier body weights than lidocaine injected animals (Fig. 1B). There was a significant main effect of sex on adult body weight (P50.0001) in both groups. For Oc1M volume, there was a significant effect of sex in the lidocaine-injected animals (P50.0001), with males having a larger Oc1M volume than females, but not in the halothane-exposed animals (P50.819) (Fig. 2A). For the volume of Oc1B, lidocaine injected males had a significantly larger volume than the lidocaine injected females (P50.0001). This was not found between the halothane exposed males and females (P50.986) (Fig. 2B). The present experiment demonstrated that halothane, a commonly used anesthetic for neonatal procedures such as gonadectomies, alters cortical morphology in rats and abolishes sex differences after a single exposure. In the
˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
123
the effects were not mediated by general metabolism or overall growth. Also, large sex differences were found in these measures in the halothane exposed rats compared to the lidocaine exposed animals. The visual cortical volumes show an entirely different pattern. The sizable sex difference seen in lidocaine exposed rats was not observed in the halothane exposed rats. The relatively minor effects on brain weight and the persistence of sex difference in brain weight indicates that halothane effects, like cryoanesthesia, are regionally specific [1,5]. It is interesting that the cerebral cortex is affected by both anesthetics. Halothane appears to have a differential effect upon the sexes, in spite of equivalent exposure time. While neonatal halothane exposure in males decreased visual cortex volume by 6–12% relative to lidocaine-injected males, halothane exposure in females increased volume 11–17% relative to lidocaine-exposed females. This study highlights the deleterious effects of halothane, a commonly used anesthetic, upon cortical morphology in neonates. A single 15-min exposure to halothane on day 1 eliminated the sex difference observed in the adult rat primary visual cortex. These data indicate that the use of any anesthetic in the neonatal rat may alter neural morphology.
Acknowledgements This work was supported by NSF IBN 9723918 to J.M.J. J.L.N. was supported by a predoctoral fellowship NIGMS 1 F31 GM17209.
References
Fig. 2. Effect of halothane versus lidocaine exposure upon the total volume of Oc1M (A) and Oc1B (B) in the adult rat. There was a significant effect of sex for both Oc1M and Oc1B in the lidocaine injected animals (P50.0001 for both measures), but not in the halothane exposed animals.
absence of any anesthesia in a prior study [9] and in the present study when neonatal rats were given only to a local injection of lidocaine, sex differences were found in the volume of the monocular (Oc1M) and binocular (Oc1B) subfields of the rat primary visual cortex. It does not appear that neonatal lidocaine injections influenced the sex difference in visual cortex volume because the present study documented a 22% difference between males and females in Oc1B, and a 19% difference was previously found in Oc1B in our lab [9]. Neonatal halothane does not appear to affect all measures in the same way or degree. Body weight was minorly (but significantly) higher, while brain weight was somewhat smaller in halothane exposed rats. This indicates that
[1] M.F. Eckenhoff, R.G. Eckenhoff, Quantitative autoradiography of halothane binding in rat brain, J. Pharmacol. Exp. Ther. 285 (1998) 371–376. [2] W.L. Hayes, Comparisons among means, in: W.L. Hayes (Ed.), Statistics, Harcourt Brace College, Fort Worth, 1994, pp. 423–471. [3] C.D. Jacobson, L.L. Antolick, R. Scholey, E. Uemura, The influence of prenatal pentobarbital exposure on the growth of dendrites in the rat hippocampus, Dev. Brain Res. 44 (1988) 233–239. ˜ [4] J.L. Nunez, B.Y. Kim, J.M. Juraska, Neonatal cryoanesthesia. affects the morphology of the visual cortex in the adult rat, Dev. Brain Res. 111 (1998) 89–99. ˜ [5] J.L. Nunez, W.A. Koss, J.M. Juraska, Hippocampal anatomy and water maze performance are affected by neonatal cyroanesthesia in rats of both sexes, Horm. Behav. 37 (2000) 169–178. ˜ [6] J.L. Nunez, J. Sodhi, J.M. Juraska, Neonatal androgens affect neuron number in the adult male and female rat visual cortex. Cereb. Cortex (in press, pending revision). [7] C.M. Park, K.E. Clegg, C.J. Harvey-Clark, M.J. Hollenberg, Improved techniques for successful neonatal rat surgery, Lab. Anim. Sci. 42 (1992) 508–513. [8] C.B. Phifer, L.M. Terry, Use of hypothermia for general anesthesia in preweanling rodents, Physiol. Behav. 38 (1986) 887–890. [9] S.N.M. Reid, J.M. Juraska, Sex differences in neuron number in the binocular area of the rat visual cortex, J. Comp. Neurol. 321 (1992) 448–455.
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˜ , J.M. Juraska / Developmental Brain Research 124 (2000) 121 – 124 J.L. Nunez
[10] H.L. Rosomoff, D.A. Holaday, Cerebral blood flow and cerebral oxygen consumption during hypothermia, Am. J Physiol. 179 (1954) 85–88. [11] E. Uemura, W.P. Ireland, E.D. Levin, R.E. Bowman, Effects of halothane on the development of rat brain: a golgi study of dendritic growth, Exp. Neurol. 89 (1985) 503–519.
[12] E. Uemura, R.E. Bowman, Effects of halothane on cerebral synaptic density, Exp. Neurol. 69 (1980) 135–142. [13] D.K. Yi, G.A. Barr, The suppression of formalin-induced c-fos expression by different anesthetic agents in the infant rat, Dev. Psychobiol. 29 (1996) 497–506.