Urologic care and progression to end-stage kidney disease: a Chronic Kidney Disease in Children (CKiD) nested case-control study.

INTRODUCTION: Children with chronic kidney disease (CKD) risk progressing to end-stage kidney disease (ESKD). The majority of CKD causes in children are related to congenital anomalies of the kidney and urinary tract, which may be treated by urologic care. OBJECTIVE: To examine the association of ESKD with urologic care in children with CKD. STUDY DESIGN: This was a nested case-control study within the Chronic Kidney Disease in Children (CKiD) prospective cohort study that included children aged 1-16 years with non-glomerular causes of CKD. The primary exposure was prior urologic referral with or without surgical intervention. Incidence density sampling matched each case of ESKD to up to three controls on duration of time from CKD onset, sex, race, age at baseline visit, and history of low birth weight. Conditional logistic regression analysis was performed to estimate rate ratios (RRs) for the incidence of ESKD. RESULTS: Sixty-six cases of ESKD were matched to 153 controls. Median age at baseline study visit was 12 years; 67% were male, and 7% were black. Median follow-up time from CKD onset was 14.9 years. Seventy percent received urologic care, including 100% of obstructive uropathy and 96% of reflux nephropathy diagnoses. Cases had worse renal function at their baseline visit and were less likely to have received prior urologic care. After adjusting for income, education, and insurance status, urology referral with surgery was associated with 50% lower risk of ESKD (RR 0.50 [95% confidence interval [CI] 0.26-0.997), compared to no prior urologic care (Figure). After excluding obstructive uropathy and reflux nephropathy diagnoses, which were highly correlated with urologic surgery, the association was attenuated (RR 0.72, 95% CI 0.24-2.18). DISCUSSION: In this study, urologic care was commonly but not uniformly provided to children with non-glomerular causes of CKD. Underlying specific diagnoses play an important role in both the risk of ESKD and potential benefits of urologic surgery. CONCLUSION: Within the CKiD cohort, children with non-glomerular causes of CKD often received urologic care. Urology referral with surgery was associated with lower risk of ESKD compared to no prior urologic care but depended on specific underlying diagnoses.

The molar tooth sign: a new Joubert syndrome and related cerebellar disorders classification system tested in Egyptian families.

Joubert syndrome and related cerebellar disorders (JSRD) are a group of recessive congenital ataxia conditions usually showing neonatal hypotonia, dysregulated breathing rhythms, oculomotor apraxia, and mental retardation. The pathognomonic finding in JSRD is the unique molar tooth sign (MTS) on brain imaging. There is a tremendously broad spectrum of signs and symptoms mainly including kidney, retina, and liver disease, along with polydactyly and facial dysmorphisms. Here we propose a new diagnostic classification within JSRD that includes four major subtypes. To test this classification, we performed a systematic recruitment and genetic evaluation from a single referral center in Egypt. Thirteen families were identified, four showed evidence of linkage to one of the four known genetic loci, three showed novel AHI1 mutations, and nine were excluded from known loci. Each family could be classified into one of the four subtypes. This classification may thus be useful in the evaluation of patients with JSRD.

Expression of the adaptor protein BLNK/SLP-65 in childhood acute lymphoblastic leukemia.

Deficient expression of BLNK, an adaptor molecule crucial for normal B-cell development, is associated with increased pro-B/pre-B-cell expansion in mice. It has been proposed that BLNK deficiency is a primary cause of B-lineage acute lymphoblastic leukemia (ALL). We studied BLNK expression in the leukemic cells from 352 patients with childhood ALL (309 B-lineage; 43 T-lineage). By HG_U95Av2 Affymetrix GeneChip analysis, BLNK was expressed in 275 of 284 (96.8%) B-lineage ALL samples but in only one of 43 (2.3%) T-lineage ALL samples. Of 118 B-lineage ALL samples analyzed with the HG_U133A GeneChip, 117 (99.2%) expressed BLNK. All 30 primary B-lineage ALL samples studied by RT-PCR expressed BLNK transcripts; all 19 samples studied by Western blotting or flow cytometry expressed BLNK protein. Levels of BLNK in B-lineage ALL were as high as those of their normal counterparts; they were not related with genetic subgroups or differentiation stage. These results indicate that BLNK deficiency is a rare occurrence in childhood B-lineage ALL and is unlikely to be a common leukemogenic event as previously proposed.

Lissencephaly with cerebellar hypoplasia (LCH): a heterogeneous group of cortical malformations.

Detailed classification of brain malformations such as lissencephaly has led to the positional cloning of genes required for normal neuronal migration and the identification of unique molecular pathways governing brain structure. While classical magnetic resonance imaging (MRI) patterns of lissencephaly involve primarily the cerebral cortex, malformations in this spectrum can be associated with significant cerebellar underdevelopment and have recently been referred to as lissencephaly with cerebellar hypoplasia (LCH). The phenotypic features of 34 children were found to define 6 subtypes of LCH. Two of these (LCHa and LCHb) were associated with mutation in the LIS1, DCX and RELN genes, respectively. Gene mutations that exemplify four additional classes (LCHc, d, e and f) remained to be determined. Phenotypic features included small head circumference, cortical malformation ranging from agyria to simplification of the gyral pattern and from near normal cortical thickness to marked thickening of the cortical gray matter. Cerebellar manifestations ranged from midline hypoplasia to diffuse volume reduction and disturbed foliation. We conclude that LCH is within the spectrum of DCX and LIS1 mutations, that LCH associated with RELN mutation is distinguished by the severity of cerebellar and hippocampal involvement, and that several distinctive patterns indicate additional genetic mutations that can produce LCH.

Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice.

Cyclooxygenase-1 (COX-1), a rate-limiting enzyme in the synthesis of prostanoids, is involved in selected vasodilatatory responses of the cerebral circulation. Cyclooxygenase-1-null mice were used to determine whether COX-1 influences cerebral ischemic damage. The middle cerebral artery was occluded in COX-1 -/- and +/+ mice (n = 9/group), and lesion volume was determined in thionin-stained sections 24 or 96 hours later. Middle cerebral artery occlusion produced larger infarcts in COX-1 -/- mice, both at 24 (35 +/- 17%; P < 0.05) and 96 hours (41 +/- 16%; P < 0.05) after ischemia. The enlargement was not due to increased susceptibility to glutamate excitotoxicity, because microinjection of N-methyl-D-aspartate or kainate in the parietal cortex produced comparable lesions in COX-1 +/+ and -/- mice ( P > 0.05; n = 8/group). To examine the contribution of hemodynamic factors to the enlargement of the infarct, cerebral blood flow was monitored by laser-Doppler flowmetry in the ischemic territory (n = 6/group). Although the reduction in cerebral blood flow was comparable in the ischemic core ( P > 0.05), at the periphery of the ischemic territory the reduction was greater in COX-1 -/- mice (-58 +/- 4%) than in COX-1 +/+ mice (-34 +/- 5%; P < 0.05). It is concluded that mice lacking COX-1 are more susceptible to focal cerebral ischemia, an effect that can be attributed to a more severe cerebral blood flow reduction in vulnerable regions at the periphery of the ischemic territory. Thus, the vascular effects of COX-1 may contribute to maintain cerebral blood flow in the postischemic brain and, as such, play a protective role in ischemic brain injury.

Cyclooxygenase-2 inhibitor ns-398 protects neuronal cultures from lipopolysaccharide-induced neurotoxicity.

BACKGROUND AND PURPOSE: The prostanoid-synthesizing enzyme cyclooxygenase (COX)-2 is markedly upregulated after cerebral ischemia and may participate in the mechanisms by which postischemic inflammation contributes to the late stages of ischemic brain injury. In the present study, we sought to provide additional evidence for a role of COX-2 in the mechanisms of neurotoxicity associated with inflammation. METHODS: Nine-day-old neuronal-glial cultures, prepared from the cerebral cortex of newborn C57BL/6J mice, were exposed to lipopolysaccharide (LPS), a potent proinflammatory agent. The contribution of COX-2 was investigated by using the COX-2 inhibitor NS-398. RESULTS: LPS produced a dose-dependent (0.001 to 10 microg/mL) and selective neuronal death that was well developed 72 hours after treatment. The effect was associated with a marked increase in the concentration of the COX reaction product prostaglandin E(2) (PGE(2)) and of the cytokine tumor necrosis factor-alpha (TNF-alpha). NS-398 (10 micromol/L) blocked the PGE(2) increase, attenuated the TNF-alpha increase, and prevented the neuronal death produced by LPS. TNF-alpha-blocking antibodies attenuated LPS-induced neuronal death, but the protection was less pronounced than that afforded by NS-398. LPS failed to elevate PGE(2) or to produce cell death in neuron-enriched cultures, suggesting that glial cells are required for these effects. CONCLUSIONS: COX-2, in part through TNF-alpha-related mechanisms, contributes to LPS-induced neuronal death. The data support the hypothesis that COX-2, in addition to its role in glutamate excitotoxicity, participates in the cytotoxicity associated with inflammation.

Human brain malformations and their lessons for neuronal migration.

The developmental steps required to build a brain have been recognized as a distinctive sequence since the turn of the twentieth century. As marking tools for experimental embryology emerged, the cellular events of cortical histogenesis have been intensively scrutinized. On this rich backdrop, molecular genetics provides the opportunity to play out the molecular programs that orchestrate these cellular events. Genetic studies of human brain malformation have proven a surprising source for finding the molecules that regulate CNS neuronal migration. These studies also serve to relate the significance of genes first identified in murine species to the more complex human brain. The known genetic repertoire that is special to neuronal migration in brain has rapidly expanded over the past five years, making this an appropriate time to take stock of the emerging picture. We do this from the perspective of human brain malformation syndromes, noting both what is now known of their genetic bases and what remains to be discovered.

Cyclooxygenase-1 participates in selected vasodilator responses of the cerebral circulation.

Cyclooxygenase (COX) is a prostanoid-synthesizing enzyme present in 2 isoforms: COX-1 and COX-2. Although it has long been hypothesized that prostanoids participate in cerebrovascular regulation, the lack of adequate pharmacological tools has led to conflicting results and has not permitted investigators to define the relative contribution of COX-1 and COX-2. We used the COX-1 inhibitor SC-560 and COX-1-null (COX-1(-/-)) mice to investigate whether COX-1 plays a role in cerebrovascular regulation. Mice were anesthetized (urethane and chloralose) and equipped with a cranial window. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry or by the (14)C-iodoantipyrine technique with quantitative autoradiography. In wild-type mice, SC-560 (25 micromol/L) reduced resting CBF by 21+/-4% and attenuated the CBF increase produced by topical application of bradykinin (-59%) or calcium ionophore A23187 (-49%) and by systemic hypercapnia (-58%) (P<0.05 to 0.01). However, SC-560 did not reduce responses to acetylcholine or the increase in somatosensory cortex blood flow produced by vibrissal stimulation. In COX-1(-/-) mice, resting CBF assessed by (14)C-iodoantipyrine was reduced (-13% to -20%) in cerebral cortex and other telencephalic regions (P<0.05). The CBF increase produced by bradykinin, A23187, and hypercapnia, but not acetylcholine or vibrissal stimulation, were attenuated (P<0.05 to 0.01). The free radical scavenger superoxide dismutase attenuated responses to bradykinin and A23187 in wild-type mice but not in COX-1(-/-) mice, suggesting that COX-1 is the source of the reactive oxygen species known to mediate these responses. The data provide evidence for a critical role of COX-1 in maintaining resting vascular tone and in selected vasodilator responses of the cerebral microcirculation.

Mutation analysis of the DCX gene and genotype/phenotype correlation in subcortical band heterotopia.

Subcortical band heterotopia (SBH) comprises part of a spectrum of phenotypes associated with classical lissencephaly (LIS). LIS and SBH are caused by alterations in at least two genes: LIS1 (PAFAH1B1) at 17p13.3 and DCX (doublecortin) at Xq22.3-q23. DCX mutations predominantly cause LIS in hemizygous males and SBH in heterozygous females, and we have evaluated several families with LIS male and SBH female siblings. In this study, we performed detailed DCX mutation analysis and genotype-phenotype correlation in a large cohort with typical SBH. We screened 26 sporadic SBH females and 11 LIS/SBH families for DCX mutations by direct sequencing. We found 29 mutations in 22 sporadic patients and 11 pedigrees, including five deletions, four nonsense mutations, 19 missense mutations and one splice donor site mutation. The DCX mutation prevalence was 84.6% (22 of 26) in sporadic SBH patients and 100% (11 of 11) in SBH pedigrees. Maternal germline mosaicism was found in one family. Significant differences in genotype were found in relation to band thickness and familial vs sporadic status.

Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice.

Cyclooxygenase-2 (COX-2), a prostanoid-synthesizing enzyme that contributes to the toxicity associated with inflammation, has recently emerged as a promising therapeutic target for several illnesses, ranging from osteoarthritis to Alzheimer's disease. Although COX-2 has also been linked to ischemic stroke, its role in the mechanisms of ischemic brain injury remains controversial. We demonstrate that COX-2-deficient mice have a significant reduction in the brain injury produced by occlusion of the middle cerebral artery. The protection can be attributed to attenuation of glutamate neurotoxicity, a critical factor in the initiation of ischemic brain injury, and to abrogation of the deleterious effects of postischemic inflammation, a process contributing to the secondary progression of the damage. Thus, COX-2 is involved in pathogenic events occurring in both the early and late stages of cerebral ischemia and may be a valuable therapeutic target for treatment of human stroke.

Stellate neurons mediate functional hyperemia in the cerebellar molecular layer.

Mice lacking cyclin D2 have a profound reduction in the number of stellate neurons in the cerebellar molecular layer. We used cyclin D2-null mice to study the contribution of stellate neurons in the increase of cerebellar blood flow (BFcrb) produced by neural activation. Crus II, a region of the cerebellar cortex that receives trigeminal sensory afferents, was activated by stimulation of the upper lip (5-30 V; 10 Hz), and BFcrb was recorded at the activated site by the use of a laser-Doppler flow probe. In wild-type mice, upper lip stimulation increased BFcrb in crus II by 32 +/- 2%. The rise in BFcrb was attenuated by 19% in heterozygous mice and by 69% in homozygous mice. In contrast to the cerebellum, the increases in somatosensory cortex blood flow produced by upper lip stimulation was not attenuated in D2-null mice. The field potentials evoked in crus II by upper lip stimulation did not differ between wild-type and D2-null mice. Stellate neurons are a major source of nitric oxide (NO) in the cerebellar molecular layer. The neuronal NO synthase inhibitor 7-nitroindazole attenuated the vascular response to crus II activation in wild-type mice but not in D2-null mice, suggesting that stellate neurons are the major source of NO mediating the vascular response. The data provide evidence that stellate neurons are a critical link between neural activity and blood flow in the activated cerebellum and that NO is the principal effector of their vascular actions.

Gene-dosing effect and persistence of reduction in ischemic brain injury in mice lacking inducible nitric oxide synthase.

We investigated whether the reduction in ischemic brain injury in inducible nitric oxide synthase (iNOS) null mice is related to the iNOS gene copy number, and whether such protection is long lasting. The middle cerebral artery (MCA) was occluded in heterozygous (+/-) and homozygous (-/-) iNOS null mice, as well as in wild-type littermates (iNOS +/+). Four days after MCA occlusion, total infarct volume was reduced by 29% in iNOS -/- mice (n=6; P<0.05) and by 14% in iNOS+/-mice (n=8; P<0.05), compared to iNOS +/+ (n=8). Ten days after MCA occlusion, total infarct volume was still reduced in iNOS +/- (-14%) and -/- mice (-21%; P<0.05 from iNOS +/+; n=8/group). These data indicate that the reduction in infarct volume is greater in iNOS -/- than in iNOS +/- mice and that the effect is stable in time. We conclude that the reduction in ischemic damage conferred by iNOS deletion exhibits a gene-dosing effect and that the protection is long lasting.

Somatic and germline mosaic mutations in the doublecortin gene are associated with variable phenotypes.

Mutations in the X-linked gene doublecortin lead to "double cortex" syndrome (DC) in females and to X-linked lissencephaly (XLIS) in males. Because most patients with DC and XLIS are sporadic, representing de novo doublecortin mutations, we considered that some of these patients could be somatic or germline mosaics. Among a population of 20 patients and their families, we found evidence for mosaic doublecortin mutations in 6 individuals. Germline mosaicism was identified in two unaffected women, each with two affected children. Additionally, one affected male with DC was found to be a somatic mosaic, which presumably spared him from the more severe phenotype of lissencephaly. The high rate of mosaicism indicates that there may be a significant recurrence risk for DC/XLIS in families at risk, even when the mother is unaffected.

Cyclooxygenase-2 contributes to functional hyperemia in whisker-barrel cortex.

The prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) is expressed in selected cerebral cortical neurons and is involved in synaptic signaling. We sought to determine whether COX-2 participates in the increase in cerebral blood flow produced by synaptic activity in the somatosensory cortex. In anesthetized mice, the vibrissae were stimulated mechanically, and cerebral blood flow was recorded in the contralateral somatosensory cortex by a laser-Doppler probe. We found that the COX-2 inhibitor NS-398 attenuates the increase in somatosensory cortex blood flow produced by vibrissal stimulation. Furthermore, the flow response was impaired in mice lacking the COX-2 gene, whereas the associated increase in whisker-barrel cortex glucose use was not affected. The increases in cerebral blood flow produced by hypercapnia, acetylcholine, or bradykinin were not attenuated by NS-398, nor did they differ between wild-type and COX-2 null mice. The findings provide evidence for a previously unrecognized role of COX-2 in the mechanisms coupling synaptic activity to neocortical blood flow and provide an insight into one of the functions of constitutive COX-2 in the CNS.

Crooked tail (Cd) models human folate-responsive neural tube defects.

Genetic correlation of human neural tube defects (NTDs) with NTD genes identified in mouse may unravel predisposing complex traits for assessment of individual risk and treatment in clinical settings. Folic acid (FA) can reduce the recurrence of NTDs in human populations by as much as 50-70%, though the mechanism of this rescue is unknown. We examined whether Crooked tail ( Cd ), a mouse strain prone to exencephaly, could provide a genetic animal model for folate-responsive NTDs. The Cd locus was localized to a 0.2 cM interval of the Mouse Genome Database genetic map, identifying tightly linked markers for genotyping prior to phenotypic expression. In a controlled diet study, Cd was found to mimic closely the clinical response to FA. FA supplementation reduced the recurrence risk of Cd exencephaly by as much as 55%. This rescue was dose dependent and did not require subjects to be inherently folate deficient. Like the female predominance of NTDs in humans, female Cd embryos were most likely to display exencephaly and were more responsive than males to the FA rescue. Importantly, FA supplementation shifted the severity of Cd phenotypic expression from early embryonic lethality to longer survival, and reduced the incidence of NTDs. The Cd locus is distinct from the known genes associated with neurulation defects, and isolation of this gene will assist identification of biochemical, genetic and gender-dependent factors contributing to folate-responsive NTDs.

The cyclooxygenase-2 inhibitor NS-398 ameliorates ischemic brain injury in wild-type mice but not in mice with deletion of the inducible nitric oxide synthase gene.

The authors investigated the role of the prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) in the mechanisms of focal cerebral ischemia and its interaction with inducible nitric oxide synthase (iNOS). Focal cerebral ischemia was produced by permanent occlusion of the middle cerebral artery (MCA) in mice. Infarct volume was measured 96 hours later by computer-assisted planimetry in thionin-stained brain sections. The highly selective COX-2 inhibitor NS398 (20 mg/kg; intraperitoneally), administered twice a day starting 6 hours after MCA occlusion, reduced total infarct volume in C57BL/6 (-23%) and 129/SVeV mice (-21%), and ameliorated the motor deficits produced by MCA occlusion (P < .05). However, NS398 did not influence infarct volume in mice with deletion of the iNOS gene (P > .05). In contrast, the neuronal NOS inhibitor 7-NI (50 mg/kg; intraperitoneally), administered once 5 minutes after MCA occlusion, reduced neocortical infarct volume by 20% in iNOS -/- mice (P < .05). NS398 did not affect arterial pressure, resting CBF or the CBF reactivity to hypercapnia in anesthetized iNOS null mice (P > .05). The data suggest that COX-2 reaction products, in mouse as in rat, contribute to ischemic brain injury. However, the failure of NS398 to reduce infarct volume in iNOS null mice suggests that iNOS-derived NO is required for the deleterious effects of COX-2 to occur. Thus, COX-2 reaction products may be another mechanism by which iNOS-derived NO contributes to ischemic brain injury.

Prevalence of dental occlusal variables and intraarticular temporomandibular disorders: molar relationship, lateral guidance, and nonworking side contacts.

STATEMENT OF PROBLEM: The association between dental occlusion and the development of intraarticular temporomandibular disorders remains unclear. PURPOSE: This study evaluated the prevalence of molar relationship, lateral guidance and nonworking side contacts and intraarticular temporomandibular disorders. MATERIAL AND METHODS: Eighty-two asymptomatic volunteers and 263 symptomatic temporomandibular disorder (TMD) patients completed a subjective questionnaire that documented the absence of jaw pain, joint noise, locking, and a positive history for TMD. Participants also underwent clinical and dental examination for signs and symptoms commonly associated with TMD or internal derangements. RESULTS: The most prevalent molar relationship was Class I. Symptomatic patients had a higher prevalence of Class II, Division 1 relationships on the left side compared with the asymptomatic volunteers with normal joints. There was a higher prevalence of canine guidance (52.04%; P <.005) on the right side in the symptomatic patients with disk displacement (DD). Volunteers with normal joints had a higher prevalence of 1 or more nonworking side contacts compared with symptomatic patients with normal joints (P <.001) and symptomatic patients with DD (P <. 001). CONCLUSION: This study suggests there are no systematic dental occlusal differences that clearly separate symptomatic from asymptomatic patients. Results indicate that it is unclear as to the relationship of the 3 analyzed factors and of intraarticular TMDs.

Differences in the gyral pattern distinguish chromosome 17-linked and X-linked lissencephaly.

BACKGROUND: Classical lissencephaly or "smooth brain" is a human brain malformation that consists of diffuse agyria and pachygyria. Two genes associated with classical lissencephaly have recently been cloned-LIS1 from chromosome 17p13.3 and XLIS (also called DCX) from Xq22.3-q23. OBJECTIVE: We performed genotype-phenotype analysis in children with lissencephaly associated with mutations of different genes. METHODS: We compared the phenotype, especially brain imaging studies, in a series of 48 children with lissencephaly, including 12 with Miller-Dieker syndrome (MDS), which is associated with large deletions of LIS1 and other genes in the region, 24 with isolated lissencephaly sequence caused by smaller LIS1 deletions or mutations, and 12 with isolated lissencephaly sequence caused by XLIS mutations. RESULTS: We found consistent differences in the gyral patterns, with the malformation more severe posteriorly in individuals with LIS1 mutations and more severe anteriorly in individuals with XLIS mutations. Thus, mutations of LIS1 are associated with a posterior-to-anterior gradient of lissencephaly, whereas mutations of XLIS are associated with an anterior-to-posterior gradient. We also confirmed differences in severity between MDS and ILS17. Hypoplasia of the cerebellar vermis proved to be more common with XLIS mutations. CONCLUSION: It is often possible to predict the gene mutation from careful review of brain imaging studies.

Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia.

The prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2) is up-regulated in the brain of rodents during cerebral ischemia and contributes to ischemic brain injury. This study sought to determine whether COX-2 is also up-regulated in the human brain in the acute stages of cerebral ischemic infarction. Paraffin-embedded sections from patients who died 1-2 days following infarction in the middle cerebral artery territory were processed for COX-2 immunohistochemistry. COX-2 immunoreactivity was observed in infiltrating neutrophils, in vascular cells and in neurons located at the border of the infarct. The data suggest that COX-2 up-regulation is also relevant to cerebral ischemia in humans and raise the possibility that COX-2 reaction products participate in the mechanisms of ischemic injury also in the human brain.

Age-dependent increase in ischemic brain injury in wild-type mice and in mice lacking the inducible nitric oxide synthase gene.

The authors investigated the influence of age on the outcome of cerebral ischemia in wild-type mice and in mice with a deletion of the inducible nitric oxide synthase (iNOS) gene. The middle cerebral artery was permanently occluded in iNOS-null mice and in wild-type (C57BL/6) controls aged 4, 8, 16, and 24 weeks. Infarct volume was determined in thionin-stained brain sections 4 days after permanent middle cerebral artery occlusion. No differences in forebrain volume were found among wild-type and iNOS-null mice at the ages studied (P > 0.05). In C57BL/6 mice (n = 5 to 6/group), neocortical infarct volume corrected for swelling was 28 +/- 5 mm3 in 4-week-old mice, 28 +/- 3 at 8 weeks, 35 +/- 4 at 16 weeks, and 37 +/- 6 at 24 weeks (mean +/- SD). iNOS-null mice (n = 5 to 6/group) had smaller infarcts than wild-type controls at all ages (P < 0.05). However, the magnitude of the reduction was greater in 4-week-old (-29% +/- 10%) or 8-week-old mice (-24% +/- 8%), than in 16-week-old (-14% +/- 10%) or 24-week-old mice (-11% +/- 6%). Neurologic deficit scores improved significantly between 24 and 96 hours in 4- and 8-week-old iNOS-null mice compared with age-matched wild-type mice (P < 0.05). However, in 16- or 24-week-old iNOS-null mice, neurologic deficits did not improve (P > 0.05). The authors conclude that in iNOS-/- and in wild-type mice, the size of the infarct produced by occlusion of the middle cerebral artery is larger in older than in younger mice. However, the reduction in infarct volume observed in iNOS-null mice is age-dependent and is greatest at 1 to 2 months of age. Therefore, age is a critical variable in studies of focal cerebral ischemic damage, both in wild-type mice and in mouse mutants.