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Postnatal lead poisoning impairs behavioral recovery following brain damage
NeuroToxicology 28 (2007) 1153–1157
Postnatal lead poisoning impairs behavioral recovery following brain damage J.S. Schneider *, E. Decamp Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States Received 27 February 2007; accepted 22 June 2007 Available online 17 August 2007
Abstract Lead is a potent environmental toxicant with well-known effects on intelligence, school achievement and behavior. Lead exposure is also associated with an increased risk of a variety of health problems including cancer, hypertension, cardiovascular disease, and renal disease. Considering the risk of hypertension, cardiovascular problems, and stroke following lead exposure, the current research assessed the extent to which postnatal exposure to environmentally relevant levels of lead could impair the recovery from a later occurring brain injury. Using a photochemical thrombotic stroke model we found that postnatal lead exposure significantly impaired post-stroke recovery of beam walking ability and proprioceptive limb placing. Considering the increased risk for hypertension and cardiovascular disease in lead-exposed humans, diminished capacity for repair or adaptation following lead exposure needs to now be examined in greater detail. # 2007 Elsevier Inc. All rights reserved. Keywords: Lead; Stroke; Behavior; Recovery of function
1. Introduction Childhood lead poisoning is a matter of grave public health concern due to well-known detrimental effects of lead on intelligence, cognition, school performance and behavior (see Lidsky and Schneider, 2003 for review). Neurobehavioral deficits associated with childhood lead poisoning may persist into adulthood and may even worsen over time (Fergusson et al., 1997; Needleman et al., 1990; Winneke et al., 1996). In addition, childhood lead poisoning can increase the risk of a variety of health problems in adulthood including cancer, hypertension, cardiovascular disease, and renal disease (Landrigan, 1990; Lin et al., 2001). Chronic lead exposure in adults has also been associated with elevated blood pressure and hypertension (Glenn et al., 2003, 2006), particularly in African Americans (Vupputuri et al., 2003), a segment of the population disproportionately affected by lead (Muntner et al., 2005).
* Corresponding author at: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, 521 JAH, Philadelphia, PA 19107, United States. Tel.: +1 215 503 0370; fax: +1 215 923 3808. E-mail address: [email protected] (J.S. Schneider). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.06.007
These factors may increase the risk of stroke in this population (Vupputuri et al., 2003). If childhood lead exposure increases the risk of developing hypertension and cardiovascular problems later in life and, as a consequence increases the risk of stroke or ischemic brain injury, it would be important to know whether prior lead exposure might affect the outcome from an ischemic brain injury. Lead has well-documented effects on a variety of physiological processes that could diminish the brain’s capacity for repair or adaptive synaptic change, including effects on neurotransmitter synthesis and release, calcium homeostasis, synaptic and dendritic morphology, neurotrophic factor expression and neural progenitor cell proliferation and neurogenesis (see Lidsky and Schneider, 2003 for review; Huang and Schneider, 2004; Schneider et al., 2004). The question posed in the current research is, can postnatal exposure to environmentally relevant levels of lead impair recovery from a later occurring brain injury? To address this question we employed a well-known photochemical model of thrombotic stroke centered in the hindlimb area of sensorimotor cortex. This model allows for reproducible infarct size and location and reproducible sensorimotor deficits with a predictable course of recovery (Brown et al., 2003; De Ryck et al., 1989).
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2. Materials and methods Male Long–Evans rats (starting at postnatal day 25) were fed either a standard laboratory diet (Purina RMH 1000 chow) or the same diet containing lead acetate (Purina RMH 1000 chow containing 1500 ppm lead acetate) for 30 days. At the end of the lead exposure period, all rats were placed on the standard diet without added lead. Drinking water was available ad libitum throughout the study. Beginning 30 days after the end of the lead exposure period, rats were assessed daily for 7 consecutive days for performance of beam walking and proprioceptive limb placing, sensorimotor behaviors considered to be sensitive to thrombotic lesion of the himdlimb area of the cortex (De Ryck et al., 1989). Proprioceptive limb placing reactions (of the hindlimb contralateral to the lesion) were assessed in hand held rats and involved separate tests for lateral and forward placing (results summed for analysis). Forward placing was tested by pushing the dorsal aspect of the rat’s paw against a table edge to stimulate limb muscles and joints; lateral placing was assessed similarly by pushing the lateral aspect of the rat’s paw against a table edge to stimulate limb muscles and joints. By supporting the rat’s chin and pointing the head 458 upward, facial contact and use of visual stimuli to influence the response were avoided. Limb placing scores were: 0 (no placing); 1 (incomplete and/or delayed placing; or 2 (immediate and complete placing). Spontaneous limb placing and more complex sensorimotor activity was assessed by the ability of rats to traverse a narrow 2.5 cm wide 2 m long beam suspended 30 cm above a table top. The number of limb placing errors made while traversing the beam were recorded for the hindlimb contralateral to the infarct. The limb ipsilateral to the infarct was tested as a control both before and after the lesion. Since ipsilateral limb use did not change at any time during the study, these data are not discussed. Animals reached an asymptotic level of performance on all measures within the 7 days of the baseline testing and were then lesioned. Photochemically induced ischemic damage to the hindlimb area of the parietal sensorimotor cortex was produced using a modification of the procedures of Watson et al. (1988). Rats were anesthetized with sodium pentobarbital (50 mg/kg), placed in a stereotaxic frame on a homeothermic blanket (to maintain constant core body temperature), the scalp incised and the location of the hindlimb sensorimotor cortex marked on the skull surface. Rose Bengal (30 mg/kg, Sigma/RBI) was then administered slowly through the tail vein. Occlusion of cortical microvessels was accomplished by exposure of the marked skull surface to an argon laser (Evergreen, Inc.) attached to a power supply, fan, optical diaphragm, focusing lens and reflective mirror mounted on an optical rail. The laser settings (514 nm wavelength) and focusing lens were adjusted to produce a 3 mm diameter beam of 200 mW. The beam was left in place on the skull surface for 5 min beginning immediately after administration of the Rose Bengal. Immediately following laser exposure, the scalp was closed and the animal was placed on a heating pad in the dark until recovery from anesthesia. Beginning 24 h after the injury, animals were tested daily for 7 days on the same behavioral measures as prior to stroke. Both
the person producing the lesion and the tester were blind to an animal’s group assignment. On the 8th day following induction of the lesion, animals were deeply anesthetized with sodium pentobarbital and euthanized by transcardial perfusion with saline followed by 4% paraformaldehyde. Brains were briefly post-fixed, sectioned at 40 mm, stained with Cresyl Violet acetate and infarct volume was calculated using a modification of a previously described method (Van Reempts et al., 1987). Briefly, the infarcted area was outlined and measured in every other Cresyl Violet-stained section using an Olympus BX-90 microscope attached to a Mac G4 computer running Image J Image Analysis Software (NIH). The volume of the infarct was then calculated by numerical integration according to the trapezoidal rule (Van Reempts et al., 1987). Blood samples were taken prior to perfusion (approximately 44 days after lead exposure) for analysis of blood lead levels by graphite furnace atomic analysis by a commercial laboratory specializing in this technique (ESA Laboratories, Inc., Chelmsford, MA). Two-way repeated measures analysis of variance was used to evaluate the effects of treatment and day on behavioral performance; a one-way analysis of variance was used to evaluate potential group differences in rate of behavioral recovery post-lesion. Infarct volumes were compared using an independent sample Student’s t-test. 3. Results Prior to injury, there were no statistically significant differences between the groups in their ability to perform any of the sensorimotor tests (Fig. 1A, B). Twenty-four hours after injury, the groups did not differ in their ability to perform beam walking but the lead-exposed group performed proprioceptive limb placing significantly worse than non lead-exposed animals (Fig. 1). For beam walking, there was a significant effect of treatment (injury or no injury, p = 0.01), time postinjury ( p < 0.001) and the interaction of treatment time post-injury ( p < 0.001). For non-lead-exposed animals, beam walking scores on days 2–7 were significantly different from scores on post-injury day 1 ( p < 0.001 for each comparison) and recovery appeared to level off at day 5. In the lead-exposed group, beam walking scores on days 3–7 were significantly different from scores on post-injury day 1 ( p < 0.05 for each comparison) and recovery appeared to level off at day 3. From days 4 to 7, the magnitude of the recovery in the lead-exposed group was significantly less than that in the non-lead group ( p < 0.01 for each comparison). Lead and non-lead-exposed groups significantly differed on proprioceptive limb placing scores over the entire post-lesion period (Fig. 1) and there was a significant effect of treatment ( p < 0.05), time post-injury ( p < 0.001) and the interaction of treatment x time post-injury ( p < 0.001). For non-leadexposed animals, proprioceptive limb placing scores on days 3–7 were significantly different from scores on post-injury day 1 ( p < 0.05 for each comparison); there was no significant effect of day on scores from the lead-exposed group ( p = 0.09). Blood lead levels were significantly different in the leadexposed (mean 18.3 mg/dl 2.9) and non lead-exposed
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Fig. 1. (A) Number of errors made on the beam walking test prior to injury (days 7 to 1) and following stroke (day 0). Lead-exposed rats (squares) and non-lead exposed controls (circles) had similar pre-lesion baseline performance and similar initial deficits. However, control animals showed significant recovery during the first week post-lesion while functional recovery in lead-exposed animals lagged significantly behind (*p < 0.01 compared to non-lead controls). (B) Rating scores on proprioceptive limb placing prior to injury (days 7 to 1) and following stroke induction (day 0). Lead-exposed rats (squares) and non-lead exposed controls (circles) had similar pre-lesion baseline performance however lead-exposed animals were significantly more impaired on this task than controls following injury. Both groups showed recovery on this task during the first week post-lesion although functional recovery in lead-exposed animals consistently lagged behind the control group (*p < 0.05 compared to non-lead controls). (C) Photograph of the typical cortical location and size of the ischemic region following photothrombotic stroke (ischemic region highlighted with red circle in photograph on right). (D) Cresyl violet-stained section illustrating the necrotic core and surrounding penumbra associated with the cortical injury (calibration = 500 mm).
(0.9 mg/dl 0.8) groups ( p < 0.01). The infarct is illustrated in Fig. 1C, D. There was no significant difference in gross infarct volume in the lead-exposed rats vs. non-lead-exposed animals (lead = 62.4 17.6 mm3; non-lead = 75.3 23.1 mm3; p > 0.05), suggesting that the lead exposure altered the response of the brain to the injury rather than magnified the extent of the injury. 4. Discussion This is the first study to show that developmental exposure to lead can negatively influence the ability of the brain to compensate for a later occurring injury. We hypothesize that the different outcomes in the groups reported in this study were due to alteration in the brain’s capacity for structural and/or functional plasticity as a consequence of lead exposure. The noninvasive photochemical lesion model has been used previously to place an infarct in a well-defined neocortical area and measure resulting neurological deficits (De Ryck et al., 1989). The model has also been used to study spontaneous recovery of function following injury (Brown et al., 2003) as well as potential neuroprotective strategies for stroke (De Ryck et al., 1996). The behaviors assessed in this study have been used by others previously and are known to have a predictable course of recovery (Brown et al., 2003). Measurement of the ability of rats to traverse a narrow elevated beam has proven to be a simple but reproducible test for quantifying the rate and
degree of a rat’s locomotor recovery after sensorimotor cortex injury and a valuable tool for studying the basic neurobiological mechansisms that may underlie recovery after stroke (Goldstein, 2003; Watson and McElligott, 1984; Yonemori et al., 1998). In the present study, we found that over the week following injury, non-lead-exposed animals showed significant improvement in beam walking whereas lead-exposed rats showed improvement on days 2 and 3 and then no further recovery. Proprioceptive limb placing is another test that has been used to assess sensorimotor integration in stroke models (Jeong et al., 2003). In the present study, recovery of this function also lagged in the lead-exposed group however, lead exposed rats also had a greater degree of impairment, compared to non-lead-exposed rats, from day 1 onward. The reasons for this are unclear at present, however, locomotor and non-locomotor sensorimotor tasks can have different responses to injury and different recovery trajectories (Jeong et al., 2003). Even though lead exposure in this study began at postnatal day 25, there are still a number of neural processes undergoing considerable changes even at this time point. In addition, although the blood brain barrier is maturing at this point, lead continues to cross the blood brain barrier after 25 days of age. Previous studies (e.g., Schneider et al., 2001) in which postweaning rats have been exposed to lead show considerable accumulation of lead in brain tissue, suggesting that there is little difficulty in lead crossing the blood brain barrier at these
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time points. Furthermore, other studies suggest that even at low concentrations, lead disrupts blood brain barrier function in weanling rats, resulting in increased brain lead levels as well as permeability to plasma proteins (e.g., Wang et al., 2007). There was approximately a 44-day time lag between the last exposure to lead and the measurement of blood lead levels. Little is known about the half-life of lead in blood in rats following chronic administration. However, based on the estimated half-life of lead in blood in humans of approximately 35 days (Lidsky and Schneider, 2003), the peak blood lead levels attained at the end of dosing may have been higher than what was currently measured. Nonetheless, the levels reported in this paper as well as the possibly higher peak levels present earlier in the study are unfortunately consistent with levels of lead poisoning seen in children in the US, according to recent Centers for Disease Control and Prevention statistics (CDC, Surveillance Data, 1997–2005). Differences in rate or extent of recovery between lead and non lead-exposed animals could not be explained by differences in infarct volumes between the groups. While we do not yet understand precisely how the neurobiological effects from lead exposure could have negatively impacted behavioral recovery from the cortical injury, there are several possible mechanisms that can be proposed. Lead impairs energy metabolism in brain, interferes with essentially all calciummediated physiological processes, interferes with neurotransmission and synapse functioning, alters dendritic branching patterns, alters gene transcription regulation (see Lidsky and Schneider, 2003 for review), decreases neurotrophic factor expression (Schneider et al., 2001) and inhibits the proliferation of neural progenitor cells (Schneider et al., 2004) as well as alters the differentiation capability and profile of progenitor cells, resulting in potentially decreased neurogenesis and increased gliosis (Huang and Schneider, 2004). While there are clearly a wide variety of cellular processes that are mobilized subsequent to a CNS injury, the possible role of newly generated cells in post-injury remodeling of the brain has only recently been appreciated (Kernie et al., 2001). For example, after a controlled cortical injury there is induction of neuronal and astrocytic populations and anatomic remodeling in large part due to proliferative replacement by neural progenitor cells (Kernie et al., 2001). While the physiological relevance of postinjury cell proliferation and differentiation is still not entirely clear, it may at least in part underlie some of the recovery that occurs post-injury (Kernie et al., 2001). Although it is not clear what role these processes may play in the time frame of the present study, it is intriguing to propose that lead exposure may compromise or alter this capacity for anatomic remodeling that may contribute to long-term impaired post-injury recovery of function. In summary, postnatal lead exposure results in impaired recovery of function following a later occurring cortical stroke injury. Considering the increased risk for hypertension and cardiovascular disease in lead-exposed humans, diminished capacity for post-injury repair or adaptation may need to be added to the long list of detrimental effects of
lead on the central nervous system. Further work is now needed in order to examine a wider variety of behaviors and study animals for longer periods of time post-lesion to examine whether lead exposure merely delays functional recovery or if lead-exposed animals permanently have worse outcomes than non-lead-exposed animals. Additional studies are also needed to know if lead exposure influences the outcome from other types of injury, such as traumatic brain injury.
References Brown AW, Marlowe KJ, Bjelke B. Age effect on motor recovery in a post-acute animal stroke model. Neurobiol Aging 2003;24:607–14. Centers for Disease Control and Prevention. Tested and Confirmed Elevated Blood Levels by State, Year and Blood Lead Level Group for Children <72 mos., CDC Surveillance Data; 1997–2005. http://www.cdc.gov/nceh/lead/ surv/stats.htm. De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Jannsen M, et al. Lubeluzole protects sensorimotor function and reduces infarct size in a photochemical stroke model in rats. J Pharmacol Exp Ther 1996;279:748–58. De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PAJ. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocoertical infarcts in rats. Stroke 1989;20:1383–90. Fergusson D, Horwood J, Lynsky M. Early dentine lead levels and educational outcomes at 18 years. J Child Psychol Psychiatr 1997;38:471–8. Glenn BS, Stewart WF, Links JM, Todd AC, Schwartz BS. The longitudinal association of lead with blood pressure. Epidemiology 2003;14:30–6. Glenn BS, Bandeen-Roche K, Lee BK, Weaver VM, Todd AC, Schwartz BS. Changes in systolic blood pressure associated with lead in blood and bone. Epidemiology 2006;17:538–44. Goldstein LB. Model of recovery of locomotor ability after sensorimotor cortex injury in rats. ILAR J 2003;44:125–9. Huang F, Schneider JS. Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain. Neurotoxicology 2004;25:1001–12. Jeong S-W, Chu K, Jung K-H, Kim SU, Kim M, Roh J-K. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke 2003;34:2258–63. Kernie SG, Erwin TM, Parada LF. Brain remodeling due to neuronal and astrocytic proliferation after controlled cortical injury in mice. J Neurosci Res 2001;66:317–26. Landrigan PJ. Current issues in the epidemiology and toxicology of occupational exposure to lead. Environ Health Perspect 1990;89:61–6. Lidsky TI, Schneider JS. Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain 2003;126:5–19. Lin JL, Tan DT, Hsu KH, Yu CC. Environmental lead exposure and progressive renal insufficiency. Arch Intern Med 2001;161:264–71. Muntner P, Menke A, DeSalvo KB, Rabito FA, Batuman V. Continued decline in blood lead levels among adults in the United States: The National Health and Nutrition Examination Surveys. Arch Intern Med 2005;165: 2155–61. Needleman HL, Schell A, Bellinger D, Allred EN. The long-term effects of exposure to low doses of lead in childhood: an 11-year follow-up report. New Engl J Med 1990;322:83–8. Schneider JS, Anderson DW, Wade TV, Decamp E, Smith MG, Zuck L, et al. Inhibition of cell proliferation in the dentate gyrus of rats following postweaning lead exposure. Neurotoxicology 2004;26:141–5. Schneider JS, Lee MH, Anderson DW, Zuck L, Lidsky TI. Enriched environment during development is protective against lead-induced neurotoxicity. Brain Res 2001;896:48–55. Van Reempts J, Van Deuren B, Vande Ven M, Cornelissen F, Borgers M. Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats. Stroke 1987;18:1113–9.
J.S. Schneider, E. Decamp / NeuroToxicology 28 (2007) 1153–1157 Vupputuri S, He J, Muntner P, Bazzano LA, Whelton PK, Batuman V. Blood lead level is associated with elevated blood pressure in Blacks. Hypertension 2003;41:463–8. Wang Q, Luo W, Zheng W, Liu Y, Xu H, Zheng G, et al. iron supplement prevents lead-induced disruption of the blood–brain barrier during rat development. Toxicol Appl Pharmacol 2007;219:33–41. Watson BD, Dietrich WD, Prado R, Green BA. Photochemically-induced vascular thrombosis (photothrombosis): central nervous system consequences and clinical possibilities. In: Gorio A, Perez-Polo JR, de Vellis J, Haber B, editors. Neural development and regeneration: cellular and
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molecular aspects NATO ASI Series, vol H22. Heidelberg: Springer Verlag; 1988. p. . p. 507–24. Watson M, McElligott JG. Cerebellar norepinephrine depletion and impaired task acquisition of specific locomotor tasks in rats.. Brain Res 1984;296: 129–38. Winneke G, Lilienthal H, Kramer U. The neurobehavioural toxicology and teratology of lead. Arch Toxicol (Suppl. 18):1996;57–70. Yonemori F, Yamaguchi T, Yamada H, Tamura A. Evaluation of a motor deficit after chronic focal cerebral ischemia in rats. J Cereb Blood Metab 1998;18:1099–106.
Postnatal lead poisoning impairs behavioral recovery following brain damage J.S. Schneider *, E. Decamp Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States Received 27 February 2007; accepted 22 June 2007 Available online 17 August 2007
Abstract Lead is a potent environmental toxicant with well-known effects on intelligence, school achievement and behavior. Lead exposure is also associated with an increased risk of a variety of health problems including cancer, hypertension, cardiovascular disease, and renal disease. Considering the risk of hypertension, cardiovascular problems, and stroke following lead exposure, the current research assessed the extent to which postnatal exposure to environmentally relevant levels of lead could impair the recovery from a later occurring brain injury. Using a photochemical thrombotic stroke model we found that postnatal lead exposure significantly impaired post-stroke recovery of beam walking ability and proprioceptive limb placing. Considering the increased risk for hypertension and cardiovascular disease in lead-exposed humans, diminished capacity for repair or adaptation following lead exposure needs to now be examined in greater detail. # 2007 Elsevier Inc. All rights reserved. Keywords: Lead; Stroke; Behavior; Recovery of function
1. Introduction Childhood lead poisoning is a matter of grave public health concern due to well-known detrimental effects of lead on intelligence, cognition, school performance and behavior (see Lidsky and Schneider, 2003 for review). Neurobehavioral deficits associated with childhood lead poisoning may persist into adulthood and may even worsen over time (Fergusson et al., 1997; Needleman et al., 1990; Winneke et al., 1996). In addition, childhood lead poisoning can increase the risk of a variety of health problems in adulthood including cancer, hypertension, cardiovascular disease, and renal disease (Landrigan, 1990; Lin et al., 2001). Chronic lead exposure in adults has also been associated with elevated blood pressure and hypertension (Glenn et al., 2003, 2006), particularly in African Americans (Vupputuri et al., 2003), a segment of the population disproportionately affected by lead (Muntner et al., 2005).
* Corresponding author at: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, 521 JAH, Philadelphia, PA 19107, United States. Tel.: +1 215 503 0370; fax: +1 215 923 3808. E-mail address: [email protected] (J.S. Schneider). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.06.007
These factors may increase the risk of stroke in this population (Vupputuri et al., 2003). If childhood lead exposure increases the risk of developing hypertension and cardiovascular problems later in life and, as a consequence increases the risk of stroke or ischemic brain injury, it would be important to know whether prior lead exposure might affect the outcome from an ischemic brain injury. Lead has well-documented effects on a variety of physiological processes that could diminish the brain’s capacity for repair or adaptive synaptic change, including effects on neurotransmitter synthesis and release, calcium homeostasis, synaptic and dendritic morphology, neurotrophic factor expression and neural progenitor cell proliferation and neurogenesis (see Lidsky and Schneider, 2003 for review; Huang and Schneider, 2004; Schneider et al., 2004). The question posed in the current research is, can postnatal exposure to environmentally relevant levels of lead impair recovery from a later occurring brain injury? To address this question we employed a well-known photochemical model of thrombotic stroke centered in the hindlimb area of sensorimotor cortex. This model allows for reproducible infarct size and location and reproducible sensorimotor deficits with a predictable course of recovery (Brown et al., 2003; De Ryck et al., 1989).
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2. Materials and methods Male Long–Evans rats (starting at postnatal day 25) were fed either a standard laboratory diet (Purina RMH 1000 chow) or the same diet containing lead acetate (Purina RMH 1000 chow containing 1500 ppm lead acetate) for 30 days. At the end of the lead exposure period, all rats were placed on the standard diet without added lead. Drinking water was available ad libitum throughout the study. Beginning 30 days after the end of the lead exposure period, rats were assessed daily for 7 consecutive days for performance of beam walking and proprioceptive limb placing, sensorimotor behaviors considered to be sensitive to thrombotic lesion of the himdlimb area of the cortex (De Ryck et al., 1989). Proprioceptive limb placing reactions (of the hindlimb contralateral to the lesion) were assessed in hand held rats and involved separate tests for lateral and forward placing (results summed for analysis). Forward placing was tested by pushing the dorsal aspect of the rat’s paw against a table edge to stimulate limb muscles and joints; lateral placing was assessed similarly by pushing the lateral aspect of the rat’s paw against a table edge to stimulate limb muscles and joints. By supporting the rat’s chin and pointing the head 458 upward, facial contact and use of visual stimuli to influence the response were avoided. Limb placing scores were: 0 (no placing); 1 (incomplete and/or delayed placing; or 2 (immediate and complete placing). Spontaneous limb placing and more complex sensorimotor activity was assessed by the ability of rats to traverse a narrow 2.5 cm wide 2 m long beam suspended 30 cm above a table top. The number of limb placing errors made while traversing the beam were recorded for the hindlimb contralateral to the infarct. The limb ipsilateral to the infarct was tested as a control both before and after the lesion. Since ipsilateral limb use did not change at any time during the study, these data are not discussed. Animals reached an asymptotic level of performance on all measures within the 7 days of the baseline testing and were then lesioned. Photochemically induced ischemic damage to the hindlimb area of the parietal sensorimotor cortex was produced using a modification of the procedures of Watson et al. (1988). Rats were anesthetized with sodium pentobarbital (50 mg/kg), placed in a stereotaxic frame on a homeothermic blanket (to maintain constant core body temperature), the scalp incised and the location of the hindlimb sensorimotor cortex marked on the skull surface. Rose Bengal (30 mg/kg, Sigma/RBI) was then administered slowly through the tail vein. Occlusion of cortical microvessels was accomplished by exposure of the marked skull surface to an argon laser (Evergreen, Inc.) attached to a power supply, fan, optical diaphragm, focusing lens and reflective mirror mounted on an optical rail. The laser settings (514 nm wavelength) and focusing lens were adjusted to produce a 3 mm diameter beam of 200 mW. The beam was left in place on the skull surface for 5 min beginning immediately after administration of the Rose Bengal. Immediately following laser exposure, the scalp was closed and the animal was placed on a heating pad in the dark until recovery from anesthesia. Beginning 24 h after the injury, animals were tested daily for 7 days on the same behavioral measures as prior to stroke. Both
the person producing the lesion and the tester were blind to an animal’s group assignment. On the 8th day following induction of the lesion, animals were deeply anesthetized with sodium pentobarbital and euthanized by transcardial perfusion with saline followed by 4% paraformaldehyde. Brains were briefly post-fixed, sectioned at 40 mm, stained with Cresyl Violet acetate and infarct volume was calculated using a modification of a previously described method (Van Reempts et al., 1987). Briefly, the infarcted area was outlined and measured in every other Cresyl Violet-stained section using an Olympus BX-90 microscope attached to a Mac G4 computer running Image J Image Analysis Software (NIH). The volume of the infarct was then calculated by numerical integration according to the trapezoidal rule (Van Reempts et al., 1987). Blood samples were taken prior to perfusion (approximately 44 days after lead exposure) for analysis of blood lead levels by graphite furnace atomic analysis by a commercial laboratory specializing in this technique (ESA Laboratories, Inc., Chelmsford, MA). Two-way repeated measures analysis of variance was used to evaluate the effects of treatment and day on behavioral performance; a one-way analysis of variance was used to evaluate potential group differences in rate of behavioral recovery post-lesion. Infarct volumes were compared using an independent sample Student’s t-test. 3. Results Prior to injury, there were no statistically significant differences between the groups in their ability to perform any of the sensorimotor tests (Fig. 1A, B). Twenty-four hours after injury, the groups did not differ in their ability to perform beam walking but the lead-exposed group performed proprioceptive limb placing significantly worse than non lead-exposed animals (Fig. 1). For beam walking, there was a significant effect of treatment (injury or no injury, p = 0.01), time postinjury ( p < 0.001) and the interaction of treatment time post-injury ( p < 0.001). For non-lead-exposed animals, beam walking scores on days 2–7 were significantly different from scores on post-injury day 1 ( p < 0.001 for each comparison) and recovery appeared to level off at day 5. In the lead-exposed group, beam walking scores on days 3–7 were significantly different from scores on post-injury day 1 ( p < 0.05 for each comparison) and recovery appeared to level off at day 3. From days 4 to 7, the magnitude of the recovery in the lead-exposed group was significantly less than that in the non-lead group ( p < 0.01 for each comparison). Lead and non-lead-exposed groups significantly differed on proprioceptive limb placing scores over the entire post-lesion period (Fig. 1) and there was a significant effect of treatment ( p < 0.05), time post-injury ( p < 0.001) and the interaction of treatment x time post-injury ( p < 0.001). For non-leadexposed animals, proprioceptive limb placing scores on days 3–7 were significantly different from scores on post-injury day 1 ( p < 0.05 for each comparison); there was no significant effect of day on scores from the lead-exposed group ( p = 0.09). Blood lead levels were significantly different in the leadexposed (mean 18.3 mg/dl 2.9) and non lead-exposed
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Fig. 1. (A) Number of errors made on the beam walking test prior to injury (days 7 to 1) and following stroke (day 0). Lead-exposed rats (squares) and non-lead exposed controls (circles) had similar pre-lesion baseline performance and similar initial deficits. However, control animals showed significant recovery during the first week post-lesion while functional recovery in lead-exposed animals lagged significantly behind (*p < 0.01 compared to non-lead controls). (B) Rating scores on proprioceptive limb placing prior to injury (days 7 to 1) and following stroke induction (day 0). Lead-exposed rats (squares) and non-lead exposed controls (circles) had similar pre-lesion baseline performance however lead-exposed animals were significantly more impaired on this task than controls following injury. Both groups showed recovery on this task during the first week post-lesion although functional recovery in lead-exposed animals consistently lagged behind the control group (*p < 0.05 compared to non-lead controls). (C) Photograph of the typical cortical location and size of the ischemic region following photothrombotic stroke (ischemic region highlighted with red circle in photograph on right). (D) Cresyl violet-stained section illustrating the necrotic core and surrounding penumbra associated with the cortical injury (calibration = 500 mm).
(0.9 mg/dl 0.8) groups ( p < 0.01). The infarct is illustrated in Fig. 1C, D. There was no significant difference in gross infarct volume in the lead-exposed rats vs. non-lead-exposed animals (lead = 62.4 17.6 mm3; non-lead = 75.3 23.1 mm3; p > 0.05), suggesting that the lead exposure altered the response of the brain to the injury rather than magnified the extent of the injury. 4. Discussion This is the first study to show that developmental exposure to lead can negatively influence the ability of the brain to compensate for a later occurring injury. We hypothesize that the different outcomes in the groups reported in this study were due to alteration in the brain’s capacity for structural and/or functional plasticity as a consequence of lead exposure. The noninvasive photochemical lesion model has been used previously to place an infarct in a well-defined neocortical area and measure resulting neurological deficits (De Ryck et al., 1989). The model has also been used to study spontaneous recovery of function following injury (Brown et al., 2003) as well as potential neuroprotective strategies for stroke (De Ryck et al., 1996). The behaviors assessed in this study have been used by others previously and are known to have a predictable course of recovery (Brown et al., 2003). Measurement of the ability of rats to traverse a narrow elevated beam has proven to be a simple but reproducible test for quantifying the rate and
degree of a rat’s locomotor recovery after sensorimotor cortex injury and a valuable tool for studying the basic neurobiological mechansisms that may underlie recovery after stroke (Goldstein, 2003; Watson and McElligott, 1984; Yonemori et al., 1998). In the present study, we found that over the week following injury, non-lead-exposed animals showed significant improvement in beam walking whereas lead-exposed rats showed improvement on days 2 and 3 and then no further recovery. Proprioceptive limb placing is another test that has been used to assess sensorimotor integration in stroke models (Jeong et al., 2003). In the present study, recovery of this function also lagged in the lead-exposed group however, lead exposed rats also had a greater degree of impairment, compared to non-lead-exposed rats, from day 1 onward. The reasons for this are unclear at present, however, locomotor and non-locomotor sensorimotor tasks can have different responses to injury and different recovery trajectories (Jeong et al., 2003). Even though lead exposure in this study began at postnatal day 25, there are still a number of neural processes undergoing considerable changes even at this time point. In addition, although the blood brain barrier is maturing at this point, lead continues to cross the blood brain barrier after 25 days of age. Previous studies (e.g., Schneider et al., 2001) in which postweaning rats have been exposed to lead show considerable accumulation of lead in brain tissue, suggesting that there is little difficulty in lead crossing the blood brain barrier at these
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time points. Furthermore, other studies suggest that even at low concentrations, lead disrupts blood brain barrier function in weanling rats, resulting in increased brain lead levels as well as permeability to plasma proteins (e.g., Wang et al., 2007). There was approximately a 44-day time lag between the last exposure to lead and the measurement of blood lead levels. Little is known about the half-life of lead in blood in rats following chronic administration. However, based on the estimated half-life of lead in blood in humans of approximately 35 days (Lidsky and Schneider, 2003), the peak blood lead levels attained at the end of dosing may have been higher than what was currently measured. Nonetheless, the levels reported in this paper as well as the possibly higher peak levels present earlier in the study are unfortunately consistent with levels of lead poisoning seen in children in the US, according to recent Centers for Disease Control and Prevention statistics (CDC, Surveillance Data, 1997–2005). Differences in rate or extent of recovery between lead and non lead-exposed animals could not be explained by differences in infarct volumes between the groups. While we do not yet understand precisely how the neurobiological effects from lead exposure could have negatively impacted behavioral recovery from the cortical injury, there are several possible mechanisms that can be proposed. Lead impairs energy metabolism in brain, interferes with essentially all calciummediated physiological processes, interferes with neurotransmission and synapse functioning, alters dendritic branching patterns, alters gene transcription regulation (see Lidsky and Schneider, 2003 for review), decreases neurotrophic factor expression (Schneider et al., 2001) and inhibits the proliferation of neural progenitor cells (Schneider et al., 2004) as well as alters the differentiation capability and profile of progenitor cells, resulting in potentially decreased neurogenesis and increased gliosis (Huang and Schneider, 2004). While there are clearly a wide variety of cellular processes that are mobilized subsequent to a CNS injury, the possible role of newly generated cells in post-injury remodeling of the brain has only recently been appreciated (Kernie et al., 2001). For example, after a controlled cortical injury there is induction of neuronal and astrocytic populations and anatomic remodeling in large part due to proliferative replacement by neural progenitor cells (Kernie et al., 2001). While the physiological relevance of postinjury cell proliferation and differentiation is still not entirely clear, it may at least in part underlie some of the recovery that occurs post-injury (Kernie et al., 2001). Although it is not clear what role these processes may play in the time frame of the present study, it is intriguing to propose that lead exposure may compromise or alter this capacity for anatomic remodeling that may contribute to long-term impaired post-injury recovery of function. In summary, postnatal lead exposure results in impaired recovery of function following a later occurring cortical stroke injury. Considering the increased risk for hypertension and cardiovascular disease in lead-exposed humans, diminished capacity for post-injury repair or adaptation may need to be added to the long list of detrimental effects of
lead on the central nervous system. Further work is now needed in order to examine a wider variety of behaviors and study animals for longer periods of time post-lesion to examine whether lead exposure merely delays functional recovery or if lead-exposed animals permanently have worse outcomes than non-lead-exposed animals. Additional studies are also needed to know if lead exposure influences the outcome from other types of injury, such as traumatic brain injury.
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