Facilitating Complex Trait Analysis via Reduced Complexity Crosses.

Genetically diverse inbred strains are frequently used in quantitative trait mapping to identify sequence variants underlying trait variation. Poor locus resolution and high genetic complexity impede variant discovery. As a solution, we explore reduced complexity crosses (RCCs) between phenotypically divergent, yet genetically similar, rodent substrains. RCCs accelerate functional variant discovery via decreasing the number of segregating variants by orders of magnitude. The simplified genetic architecture of RCCs often permit immediate identification of causal variants or rapid fine-mapping of broad loci to smaller intervals. Whole-genome sequences of substrains make RCCs possible by supporting the development of array- and targeted sequencing-based genotyping platforms, coupled with rapid genome editing for variant validation. In summary, RCCs enhance discovery-based genetics of complex traits.

Commentary to Brain-wide continuous functional ultrasound imaging for real-time monitoring of hemodynamics during ischemic stroke.

Functional ultrasound (FUS) has emerged as a novel imaging method to reliably assess relative cerebral blood volume (rCBV) and infer perfusion, with good spatiotemporal resolution. Brunner and colleagues provide what appears to be its first application to characterize peri-infarct spreading depolarizations (SDs) in experimental stroke through recording of transient hyperemic events. They also report incomplete overlap between acute perfusion deficits and subsequent infarct distribution, specifically noting a rostral expansion to involve penumbral territory from which propagating depolarizations had preferentially originated. This observation would not be straightforward using other methodologies. Other strengths and limitations of the study are considered.

Impact of C57BL/6 substrain on sex-dependent differences in mouse stroke models.

We have recently found significant variation in stroke vulnerability among substrains of C57BL/6 mice, observing that commonly used N-lineage substrains exhibit larger infarcts than C57BL/6J and related substrains. Parallel variation was also seen with respect to sex differences in stroke vulnerability, in that C57BL/6 mice of the N-lineage exhibited comparable infarct sizes in males and females, whereas infarcts tended to be smaller in females than in males of J-lineage substrains. This adds to the growing list of recognized phenotypic and genetic differences among C57BL/6 substrains. Although no previous studies have explicitly compared substrains with respect to sex differences in stroke vulnerability, unrecognized background mismatch has occurred in some studies involving control and genetically modified mice. The aims of this review are to: present the evidence for associated substrain- and sex-dependent differences in a mouse permanent occlusion stroke model; examine the extent to which the published literature in other models compares with these recent results; and consider the potential impact of unrecognized heterogeneity in substrain background on the interpretation of studies investigating the impact of genetic modifications on sex differences in stroke outcome. Substrain emerges as a critical variable to be documented in any experimental stroke study in mice.

Substrain- and sex-dependent differences in stroke vulnerability in C57BL/6 mice.

The C57BL/6 mouse strain is represented by distinct substrains, increasingly recognized to differ genetically and phenotypically. The current study compared stroke vulnerability among C57BL/6 J (J), C57BL/6JEiJ (JEiJ), C57BL/6ByJ (ByJ), C57BL/6NCrl (NCrl), C57BL/6NJ (NJ) and C57BL/6NTac (NTac) substrains, using a model of permanent distal middle cerebral artery and common carotid artery occlusion. Mean infarct volume was nearly two-fold smaller in J, JEiJ and ByJ substrains relative to NCrl, NJ and NTac (N-lineage) mice. This identifies a previously unrecognized confound in stroke studies involving genetically modified strain comparisons if control substrain background were not rigorously matched. Mean infarct size was smaller in females of J and ByJ substrains than in the corresponding males, but there was no sex difference for NCrl and NJ mice. A higher proportion of small infarcts in J and ByJ substrains was largely responsible for both substrain- and sex-dependent differences. These could not be straightforwardly explained by variations in posterior communicating artery patency, MCA anatomy or acute penumbral blood flow deficits. Their larger and more homogeneously distributed infarcts, together with their established use as the common background for many genetically modified strains, may make N-lineage C57BL/6 substrains the preferred choice for future studies in experimental stroke.

Temporal thresholds for infarction and hypothermic protection in Long-Evans rats: factors affecting apparent 'reperfusion injury' after transient focal ischemia.

BACKGROUND AND PURPOSE: Some previous studies in Long-Evans rats noted larger infarcts after transient middle cerebral artery (MCA) occlusions than after permanent occlusions, interpreted to demonstrate "reperfusion injury." Recent experiments failed to reproduce this phenomenon, prompting an investigation of the sources of variability in this animal model. METHODS: Male Long-Evans rats were subjected to surgical occlusion of the right MCA and ipsilateral common carotid artery. Variables tested included duration of occlusion and halothane anesthesia exposure and targeting of proximal or distal MCA occlusion sites. The temporal window for hypothermic protection was also investigated. RESULTS: MCA occlusions at the level of the rhinal fissure produced graded increases in infarct volume with ischemia duration, and lesion size did not differ between 3-hour and permanent occlusions independent of anesthesia duration. Occlusions at a more distal site produced infarcts of comparable size after transient 3-hour occlusions and after permanent occlusions accompanied by prolonged anesthesia, but significantly smaller infarcts were seen when permanent occlusions were followed by rapid anesthesia termination. Hypothermia conferred protection only when initiated before reperfusion after transient proximal occlusions. CONCLUSIONS: These results indicate that previously described "reperfusion injury" after transient MCA occlusions conversely reflects unexpected injury reduction when rats with permanent occlusions experience early anesthesia termination. More rapid blood pressure recovery under such conditions permits improved collateral perfusion. The absence of a detectable postischemic window for hypothermic protection further argues against a significant component of delayed postreperfusion injury in this model.

Transient cooling during early reperfusion attenuates delayed edema and infarct progression in the Spontaneously Hypertensive Rat. Distribution and time course of regional brain temperature change in a model of postischemic hypothermic protection.

The temperature threshold for protection by brief postischemic cooling was evaluated in a model of transient focal ischemia in the Spontaneously Hypertensive Rat, using an array of epidural probes to monitor regional brain temperatures. Rats were subjected to 90 mins tandem occlusion of the right middle cerebral artery (MCA) and common carotid artery. Systemic cooling to 32 degrees C was initiated 5 mins before recirculation, with simultaneous brain cooling to temperatures ranging from 28 degrees C to 32 degrees C within the MCA territory by means of a temperature-controlled saline drip. Rewarming was initiated at 2 h recirculation and was complete within 30 mins. Tissue damage and edema volume showed clear temperature-dependent reductions when evaluated at 3 days survival, with no protection evident in the group at 32 degrees C but progressive effects on both parameters after deeper cooling. A particularly striking effect was the essentially complete elimination of edema progression between 1 and 3 days. Temperature at distal sites within the MCA territory better predicted reductions in lesion volume, indicating that protection required effective cooling of the penumbral regions destined to be spared. These results show that even brief cooling can be highly protective when initiated at the time of recirculation after focal ischemia, but indicate a substantially lower temperature threshold for hypothermic protection than has been reported for other strains, occlusion methods, and cooling durations.

CBF changes associated with focal ischemic preconditioning in the spontaneously hypertensive rat.

Experimental stroke models exhibit robust protection after prior preconditioning (PC) insults. This study comprehensively examined cerebral blood flow (CBF) responses to permanent middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats preconditioned by noninjurious transient focal ischemia, using [(14)C]iodoantipyrine autoradiography at varied occlusion intervals. Preconditioning was produced by 10-min occlusion of the MCA and ipsilateral common carotid artery under halothane anesthesia. These vessels were permanently coagulated 24 h later in naïve, PC, and sham-operated rats. Infarct volumes were determined from hematoxylin-eosin-stained frozen sections after 1 or 3 days. Edema-corrected infarct volume was reduced from 127+/-21 in naïve rats to 101+/-31 and 52+/-28 mm(3) in sham and PC groups, respectively, at 1 day, with similar results at 3 days. All animals exhibited a consistent CBF threshold for infarction (approximately 30 mL/100 g/min). Tissue volumes below this threshold were identical in naïve and PC groups after 15-min occlusion. However, by 3 h the volume of ischemic cortex decreased in the PC group but remained unchanged in naïve rats, predicting final infarct volumes. Cerebral blood flow recovery was confirmed in brains of individual rats evaluated by repeated laser Doppler perfusion imaging during the same 3-h interval. Modest sham protection correlated with better-maintained global perfusion, detectable also in the contralateral cortex, apparently reflecting the PC effects of prior anesthesia. These results establish that timely reperfusion of penumbra, achieved by synergistic mechanisms, is a primary determinant of PC-induced protection in experimental stroke.

Selective, reversible occlusion of the middle cerebral artery in rats by an intraluminal approach. Optimized filament design and methodology.

These studies optimized design and application of an intraluminal filament method to achieve selective middle cerebral artery (MCA) occlusion in rats. Silicone plugs of 300 microm diameter and 700-800 microm length were molded onto 6-0 suture. These were introduced into Wistar rats previously fitted with telemetric probes, using established placement procedures, with and without heparinization. Temperature and activity were monitored for 3 days, after which lesion volumes were assessed by triphenyltetrazolium chloride staining. Optimized filaments entered the MCA in 85% of Wistar rats, failures being attributable to anatomical variation at its origin from the internal carotid artery. Infarcts restricted to the MCA territory were apparent after 90 min occlusion, and maximal after 3 h occlusion. Intraischemic hyperthermia was noted in a third of occlusions performed without heparin, but never with anticoagulant treatment. Permanent occlusions were also evaluated in Fisher, Lewis, Long-Evans, Spontaneously Hypertensive and Sprague-Dawley rats, and Wistar rats from a second supplier, and compared with data for surgical MCA occlusions. Success rates varied among strains, but infarct volumes correlated with those obtained after surgical occlusions in respective populations. These studies demonstrate the feasibility and limitations of reversible and selective intraluminal filament occlusion of the MCA in rats.

Cerebral blood flow thresholds for mRNA synthesis after focal ischemia and the effect of MK-801.

BACKGROUND AND PURPOSE: MK-801 is a noncompetitive antagonist of N-methyl-d-aspartate subtype glutamate receptors with protective efficacy in experimental stroke. This study examined the impact of MK-801 on cerebral blood flow (CBF) and its relationship to gene expression changes during focal ischemia. METHODS: Spontaneously hypertensive rats were subjected to surgical occlusion of the middle cerebral artery and ipsilateral common carotid artery after 30 minutes pretreatment with 5 mg/kg MK-801 or saline vehicle. After 2.5 hours of ischemia, regional CBF was evaluated by [14C]iodoantipyrine autoradiography and compared with distributions of gene expression changes evaluated by in situ hybridization detection of mRNAs encoding several immediate-early genes and the stress protein, hsp72. RESULTS: MK-801 increased CBF in contralateral cortex from 93+/-15 to 187+/-37 mL/100 g per minute and produced a significant 25% reduction in the volume of ischemic cortex ipsilateral to occlusion. The extent of cortex failing to express inducible mRNAs correspondingly decreased, but the CBF threshold for mRNA synthesis remained unchanged (25 to 30 mL/100 g per minute). Widespread immediate-early gene expression in the neocortex became restricted to periinfarct regions after MK-801 treatment, and hybridization patterns in the striatum and hippocampus reflected the altered topography of cortical activation after drug treatment. CONCLUSIONS: MK-801 alters ischemia-induced gene expression by 2 distinct mechanisms. Generalized increases in CBF reduce the volume of cortex falling below ischemic injury thresholds, protecting tissue and facilitating transcription of inducible genes proximal to the ischemic focus. In addition, MK-801 attenuates the signals that induce expression of immediate-early genes in cortical and subcortical regions remote from the middle cerebral artery territory.

Protective preconditioning by transient global ischemia in the rat: components of delayed injury progression and lasting protection distinguished by comparisons of depolarization thresholds for cell loss at long survival times.

Robust ischemic preconditioning has been shown in rodent brain, but there are concerns regarding the persistence of neuron protection. This issue was examined in rat hippocampus following 4-vessel occlusion (4-VO) ischemia, using DC shifts characteristic of ischemic depolarization to reproducibly define insult severity. Preconditioning ischemia producing 2 to 3.5 mins depolarization was followed at intervals of 2, 5, or 7 days by test insults of varied duration, after which CA1 counts were obtained at 1, 2, 4, or 12 weeks. Neuron loss in naive animals increased with depolarization time longer than 4 mins regardless of postischemic survival interval. Preconditioning 2, 5, or 7 days before test insults prolonged the injury threshold evaluated at 1 week survival to 15, 9, or 6 mins, respectively, showing robust protection and a rapid decay of the protected state. However, by 2 weeks survival after preconditioning at a 2-day interval, the injury threshold dramatically regressed from 15 to 9 mins. Thereafter protection remained relatively stable through 1 month, but slight progression of neuron injury was evident at 3 months. Inflammatory responses were seen in both naive and preconditioned hippocampi throughout this interval, appropriate to the extent of neuron injury. These studies show distinct components of transient and lasting protection after ischemic preconditioning. Finally, it was found that ischemic depolarization was delayed by approximately 1 min in optimally preconditioned rat hippocampus, in contrast to previous results in the gerbil, identifying one specific mechanism by which insult severity is reduced in this model.

Preconditioning cortical lesions reduce the incidence of peri-infarct depolarizations during focal ischemia in the Spontaneously Hypertensive Rat: interaction with prior anesthesia and the impact of hyperglycemia.

The relationship between peri-infarct depolarizations (PIDs) and infarction was investigated in a model of preconditioning by cortical freeze lesions (cryogenic lesions, CL) in the Spontaneously Hypertensive Rat. Small (< 5 mm(3)) lesions produced 24 hours before permanent focal ischemia were protective, without impacting baseline cerebral blood flow (CBF) and metabolism. Prior CL reduced infarct volume, associated with improved penumbral CBF as previously showed for ischemic preconditioning. The brief initial procedure avoided sham effects on infarct volume after subsequent occlusion under brief anesthesia. However, under prolonged isoflurane anesthesia for perfusion monitoring both sham and CL rats showed reduced PID incidence relative to naive animals. This anesthesia effect could be eliminated by using α-chloralose during perfusion imaging. As an additional methodological concern, blood glucose was frequently elevated at the time of the second surgery, reflecting buprenorphine-induced pica and other undefined mechanisms. Even modest hyperglycemia (>10 mmol/L) reduced PID incidence. In normoglycemic animals CL preconditioning reduced PID number by 50%, demonstrating associated effects on PID incidence, penumbral perfusion, and infarct progression. Hyperglycemia suppressed PIDs without affecting the relationship between CBF and infarction. This suggests that the primary effect of preconditioning is to improve penumbral perfusion, which in turn impacts PID incidence and infarct size.

Pharmacological manipulations of ATP-dependent potassium channels and adenosine A1 receptors do not impact hippocampal ischemic preconditioning in vivo: evidence in a highly quantitative gerbil model.

Ischemic preconditioning models have been characterized in brain, heart, and other tissues, and previous pharmacologic studies have suggested an involvement of adenosine and ATP dependent potassium (KATP) channels in such tolerance phenomena. This question was reexamined in a reproducible gerbil model in which the duration of ischemic depolarization defined the severity of preconditioning and test insults. Agents studied were glibenclamide, a blocker of KATP channels; 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist; and N6-cyclopentyladenosine (CPA), an A1 agonist. Intraventricular glibenclamide injections aggravated neuron damage after brief priming insults, in parallel with a dose-dependent prolongation of ischemic depolarization. However, the depolarization thresholds for ischemic neuronal injury were identical in vehicle- and glibenclamide-treated animals, and glibenclamide did not affect preconditioning when equivalent insult severity was maintained during priming insults. Neither DPCPX nor CPA had any effect on the onset or duration of depolarization after intraperitoneal injection in this model, and neither drug affected neuron damage. In the case of CPA, it was necessary to maintain temperature for 4 to 6 hours of recirculation to avoid significant confounding hypothermia. These results fail to support a direct involvement of A1 receptors or KATP channels during early stages in the development of ischemic tolerance in vivo, and emphasize the need for robust, well-controlled, and quantitative models in such studies.

Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning.

The distribution and time course of expression of the heat shock/stress proteins, hsp27 and hsp72, were evaluated in a highly controlled gerbil model of ischemic injury and tolerance induction, in which the duration of ischemic depolarization in each hippocampus provides a precise quantitative index of insult severity. Gerbils were subjected to brief priming insults (2- to 3.5-minute depolarization) that produce optimal preconditioning, to severe test insults (6- to 8.5-minute depolarization) that produce complete CA1 neuron loss in naive animals, or to combined insults administered 1 week apart, after which almost complete tolerance to CA1 neuron injury is observed. Immunoreactivities of hsp27, hsp72, glial fibrillary acidic protein and microtubule-associated protein 2 (MAP2) were evaluated in animals perfused at defined intervals after the final insult in each treatment group, using a variation of established antigen-retrieval procedures that significantly improves detection of many proteins in vibratome brain sections. Hsp72 was detected in CA1 neurons of some hippocampi 2 to 4 days after preconditioning, but this was only seen after the longest priming depolarizations, whereas shorter insults that still induced optimal tolerance failed to induce hsp72. Hsp72 was induced after test insults in preconditioned hippocampi, but at a higher depolarization threshold than observed for naive animals. An astrocytic localization of hsp27 was observed in regions of neuron injury, as indicated by reduced MAP2 immunoreactivity, and was primarily restricted to dentate hilus after preconditioning insults. These results establish that limited hilar lesions are characteristic of optimal preconditioning, whereas prior neuronal expression of either hsp72 or hsp27 is not required for ischemic tolerance.

Induced hippocampal neuron protection in an optimized gerbil ischemia model: insult thresholds for tolerance induction and altered gene expression defined by ischemic depolarization.

Preconditioning of hippocampal CA1 neurons was evaluated in a gerbil model of transient global ischemia using extracellular recording of DC potential shifts characteristic of ischemic depolarization to precisely define the duration of both priming and test insults. Brief ischemia resulting in depolarizations of 2.5 to 3.5 minutes consistently induced maximal tolerance (95% protection) against subsequent challenges 2 days later with an approximate doubling of the insult duration required for complete CA1 neuron loss from 6 to 12 minutes depolarization when evaluated 1 week after the test insult. Significant protection persisted at 2 months survival, although the apparent injury threshold regressed to approximately 8 minutes, indicating delayed progression of injury after longer test insults. In situ hybridization was used to evaluate depolarization thresholds for induction of mRNAs encoding the 70 kDa heat shock/stress protein, hsp72, as well as several immediate-early genes (c-fos, c-jun, junB, and junD). Immediate-early genes were prominently expressed after short insults inducing tolerance, whereas appreciable hsp72 induction only occurred after insults approaching the threshold for neuron injury. These results establish an ischemic preconditioning model with the predictability needed for mechanistic studies and demonstrate that prior transcriptional activation of the postischemic heat shock response is not required for expression of delayed tolerance.

Hypothermic protection in rat focal ischemia models: strain differences and relevance to "reperfusion injury".

Hypothermic protection was compared in Long-Evans and spontaneously hypertensive rat (SHR) strains using transient focal ischemia, and in Wistar and SHR strains using permanent focal ischemia. Focal ischemia was produced by distal surgical occlusion of the middle cerebral artery and tandem occlusion of the ipsilateral common carotid artery (MCA/CCAO). Moderate hypothermia of 2 hours' duration was produced by systemic cooling to 32 degrees C, with further cooling of the brain achieved by reducing to 30 degrees C the temperature of the saline drip superfusing the exposed occlusion site. Infarct volume was determined from serial hematoxylin and eosin-stained frozen sections obtained routinely at 24 hours, or in some cases after 3 days' survival. In the SHR, moderate hypothermia was only effective when initiated before recirculation after a 90-minute occlusion period. In contrast, the same intervention was strikingly effective in the Long-Evans rat even when initiated after as long as 30-minute reperfusion after a 3-hour occlusion. This magnitude and duration of cooling was not protective in permanent MCA/CCAO in the SHR, but such transient hypothermia did effectively reduce infarct volume after permanent occlusions in Wistar rats. These results show striking differences in the temporal window for hypothermic protection among rat focal ischemia models. As expected, "reperfusion injury" in the Long-Evans strain is particularly responsive to delayed cooling. The finding that the SHR can be protected by hypothermia initiated immediately before recirculation suggests a rapidly evolving component of injury occurs subsequent to reperfusion in this model as well. Hypothermic protection after permanent occlusion in Wistar rats identifies a transient, temperature-sensitive phase of infarct evolution that is not evident in the unreperfused SHR. These observations confirm that distinct mechanisms can underlie the temporal progression of injury in rat stroke models, and emphasize the critical importance of considering model and strain differences in extrapolating results of hypothermic protection studies in animals to the design of interventions in clinical stroke.

Depolarization thresholds for hippocampal damage, ischemic preconditioning, and changes in gene expression after global ischemia in the rat.

Induced ischemic tolerance in rat hippocampus was investigated in a forebrain ischemia model of repeated 4-vessel occlusion (4-VO). Ischemic insult variability was reduced by the use of dc potential measurements to determine the duration of ischemic depolarization in hippocampus. The results demonstrate a depolarization threshold for ischemic injury to CA1 neurons of 4-6 min and a window for optimal preconditioning of 2.5-3.5 min. Levels of induced mRNAs encoding hsp72 and several immediate-early genes were also shown to vary with depolarization interval. Immediate-early genes were maximally induced after depolarization periods inducing optimal preconditioning, while hsp72 expression increased with insult severity over the range leading to neuron loss. These results are similar to those obtained in gerbil studies indicating that preconditioning does not require large increases in hsp72 expression, and demonstrate the fundamental comparability of rodent global ischemia models when monitored by this approach.

Metabolic and perfusion responses to recurrent peri-infarct depolarization during focal ischemia in the Spontaneously Hypertensive Rat: dominant contribution of sporadic CBF decrements to infarct expansion.

Peri-infarct depolarizations (PIDs) contribute to the evolution of focal ischemic lesions. Proposed mechanisms include both increased metabolic demand under conditions of attenuated perfusion and overt vasoconstrictive responses to depolarization. The present studies investigated the relative contributions of metabolic and perfusion effects to PID-associated infarct expansion during middle cerebral artery (MCA) occlusion in the Spontaneously Hypertensive Rat. The initial distribution of ischemic depolarization (ID) was established within minutes after MCA occlusion at a cerebral blood flow threshold of ∼40 mL/100 g per minute, with expansion of the depolarized territory during 3 hours detected in half of the animals. Peri-infarct depolarizations were associated with transient metabolic responses, comparable to those observed after spreading depression, with no evidence of cumulative energy failure after multiple transient depolarizations during 1 hour. Speckle contrast imaging of PID-associated flow transients documented prominent distal hyperemic flow responses that became progressively attenuated in regions of already impaired perfusion, with modest propagated flow decreases more proximal to the ischemic core. However, sporadic PIDs were associated with persistent decrements in perfusion, increasing tissue volume below the threshold for energy failure, ID and infarction. These latter, comparatively rare, events can account for the pattern of stepwise infarct expansion in this model.

Hypothalamic thyrotropin-releasing hormone mRNA responses to hypothyroxinemia induced by sleep deprivation.

Sleep deprivation in rats results in progressive declines in circulating concentrations of both total and free thyroxine (T(4)) and triiodothyronine (T(3)) without an expected increase in plasma thyroid-stimulating hormone (TSH). Administration of thyrotropin-releasing hormone (TRH) results in appropriate increases in plasma TSH, free T(4), and free T(3) across experimental days, suggesting deficient endogenous TRH production and/or release. This study examined transcriptional responses related to TRH regulation following sleep deprivation. In situ hybridization was used to detect and quantitate expression of mRNAs encoding prepro-TRH and 5'-deiodinase type II (5'-DII) in brain sections of six rats sleep deprived for 16-21 days, when there was marked hypothyroxinemia, and in sections from animals yoked to the experimental protocol as well as from sham controls. TRH transcript levels in the paraventricular nucleus (PVN) were essentially unchanged at 15-16 days but increased to about threefold control levels in three of four rats sleep deprived for 20-21 days, a change comparable to that typically found in prolonged experimental hypothyroidism. There was no evidence for suppression of 5'-DII mRNA levels, which would be a sign of T(3) feedback downregulation of neurons in the PVN. A failure to increase serum TSH in response to hypothyroxinemia and to increased prepro-TRH mRNA expression indicates that alterations in posttranscriptional stages of TRH synthesis, processing, or release likely mediate the central hypothyroidism induced by sleep deprivation.