The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8 -10 mM) superfusate [K ϩ ], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel "on-line histology" based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0 -4. At physiological superfusate ] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure-function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K ϩ ].

The discovery of the rhythmogenic pre-Bötzinger complex (preBötC) inspiratory network, which remains active in a transverse brainstem slice, greatly increased the understanding of neural respiratory control. However, basic questions remain unanswered such as (1) What are the necessary and sufficient slice boundaries for a functional preBötC? (2) Is the minimal preBötC capable of reconfiguring between inspiratory-related patterns (e.g., fictive eupnea and sighs)? (3) How is preBötC activity affected by surrounding structures? Using newborn rat slices with systematically varied dimensions in physiological [K ϩ ] (3 mM), we found that a 175 m thickness is sufficient for generating inspiratory-related rhythms. In 700-m-thick slices with unilaterally exposed preBötC, a kernel Ͻ100 m thick, centered 0.5 mm caudal to the facial nucleus, is necessary for rhythm generation. Slices containing this kernel plus caudal structures produced eupneic bursts of regular amplitude, whereas this kernel plus rostral tissue generated sighs, intermingled with eupneic bursts of variable amplitude ("eupnea-sigh pattern"). After spontaneous arrest of rhythm, substance-P or neurokinin-1 (NK1) receptor agonist induced the eupnea-sigh burst pattern in Ն250-m-thick slices, whereas thyrotropin-releasing hormone or phosphodiesterase-4 blockers evoked the eupnea burst pattern. Endogenous rhythm was depressed by NK1 receptor antagonism. Multineuronal Ca 2ϩ imaging revealed that preBötC neurons reconfigure between eupnea and eupnea-sigh burst patterns. We hypothesize a (gradient-like) spatiochemical organization of regions adjacent to the preBötC, such that a small preBötC inspiratory-related oscillator generates eupnea under the dominant influence of caudal structures or thyrotropin-releasing hormone-like transmitters but eupnea-sigh activity when the influence of rostral structures or substance-P-like transmitters predominates.

Collateral circulation is a key variable determining prognosis and response to recanalization therapy during acute ischemic stroke. Remote ischemic perconditioning (RIPerC) involves inducing peripheral ischemia (typically in the limbs) during stroke and may reduce perfusion deficits and brain damage due to cerebral ischemia. In this study, we directly investigated pial collateral flow augmentation due to RIPerC during distal middle cerebral artery occlusion (MCAo) in rats. Blood flow through pial collaterals between the anterior cerebral artery (ACA) and the MCA was assessed in male Sprague Dawley rats using in vivo laser speckle contrast imaging (LSCI) and two photon laser scanning microscopy (TPLSM) during distal MCAo. LSCI and TPLSM revealed that RIPerC augmented collateral flow into distal MCA segments. Notably, while control rats exhibited an initial dilation followed by a progressive narrowing of pial arterioles 60 to 150-min post-MCAo (constricting to 80-90% of post-MCAo peak diameter), this constriction was prevented or reversed by RIPerC (such that vessel diameters increased to 105-110% of post-MCAo, pre-RIPerC diameter). RIPerC significantly reduced early ischemic damage measured 6 h after stroke onset. Thus, prevention of collateral collapse via RIPerC is neuroprotective and may facilitate other protective or recanalization therapies by improving blood flow in penumbral tissue.

Cerebral collateral circulation and age are critical factors in determining outcome from acute ischemic stroke. Aging may lead to rarefaction of cerebral collaterals, and thereby accelerate ischemic injury by reducing penumbral blood flow. Dynamic changes in pial collaterals after onset of cerebral ischemia may vary with age but have not been extensively studied. Here, laser speckle contrast imaging (LSCI) and two-photon laser scanning microscopy (TPLSM) were combined to monitor cerebral pial collaterals between the anterior cerebral artery (ACA) and the middle cerebral artery (MCA) in young adult and aged male Sprague Dawley rats during distal middle cerebral artery occlusion (dMCAo). Histological analysis showed that aged rats had significantly greater volumes of ischemic damage than young rats. LSCI showed that cerebral collateral perfusion declined over time after stroke in aged and young rats, and that this decline was significantly greater in aged rats. TPLSM demonstrated that pial arterioles narrowed faster after dMCAo in aged rats compared to young adult rats. Notably, while arteriole vessel narrowing was comparable 4.5 h after ischemic onset in aged and young adult rats, red blood cell velocity was stable in young adults but declined over time in aged rats. Overall, red blood cell flux through pial arterioles was significantly reduced at all time-points after 90 min post-dMCAo in aged rats relative to young adult rats. Thus, collateral failure is more severe in aged rats with significantly impaired pial collateral dynamics (reduced diameter, red blood cell velocity, and red blood cell flux) relative to young adult rats.

No abstract

circulation through cerebral collaterals can maintain tissue viability until reperfusion is achieved. However, collateral circulation is time limited, and failure of collaterals is accelerated in the aged. Remote ischemic perconditioning (RIPerC), which involves inducing a series of repetitive, transient peripheral cycles of ischemia and reperfusion at a site remote to the brain during cerebral ischemia, may be neuroprotective and can prevent collateral failure in young adult rats. Here, we demonstrate the efficacy of RIPerC to improve blood flow through collaterals in aged (16-18 months of age) Sprague Dawley rats during a distal middle cerebral artery occlusion. Laser speckle contrast imaging and two-photon laser scanning microscopy were used to directly measure flow through collateral connections to ischemic tissue. Consistent with studies in young adult rats, RIPerC enhanced collateral flow by preventing the stroke-induced narrowing of pial arterioles during ischemia. This improved flow was associated with reduced early ischemic damage in RIPerC treated aged rats relative to controls. Thus, RIPerC is an easily administered, non-invasive neuroprotective strategy that can improve penumbral blood flow via collaterals. Enhanced collateral flow supports further investigation as an adjuvant therapy to recanalization therapy and a protective treatment to maintain tissue viability prior to reperfusion.

Iron chelators, such as the intracellular ferrous chelator 2,2'-bipyridine, are a potential means of ameliorating iron-induced injury after intracerebral hemorrhage (ICH). We evaluated bipyridine against the collagenase and whole-blood ICH models and a simplified model of iron-induced damage involving a striatal injection of FeCl2 in adult rats. First, we assessed whether bipyridine (25 mg/kg beginning 12 h post-ICH and every 12 h for 3 days) would attenuate non-heme iron levels in the brain and lessen behavioral impairments (neurological deficit scale, corner turn test, and horizontal ladder) 7 days after collagenase-induced ICH. Second, we evaluated bipyridine (20 mg/kg beginning 6 h post-ICH and then every 24 h) on edema 3 days after collagenase infusion. Body temperature was continually recorded in a subset of these rats beginning 24 h prior to ICH until euthanasia. Third, bipyridine was administered (as per experiment 2) after whole-blood infusion to examine tissue loss, neuronal degeneration, and behavioral impairments at 7 days post-stroke, as well as body temperature for 3 days post-stroke. Finally, we evaluated whether bipyridine (25 mg/kg given 2 h prior to surgery and then every 12 h for 3 days) lessens tissue loss, neuronal death, and behavioral deficits after striatal FeCl2 injection. Bipyridine caused a significant hypothermic effect (maximum drop to 34.6 °C for 2-5 h after each injection) in both ICH models; however, in all experiments bipyridine-treated rats were indistinguishable from vehicle controls on all other measures (e.g., tissue loss, behavioral impairments, etc.). These results do not support the use of bipyridine against ICH.

Studies treating intracerebral hemorrhage (ICH) with therapeutic hypothermia (TH) have shown inconsistent benefits. We hypothesized that TH's anti-inflammatory effects may be responsible as inflammatory cells are essential for removing degrading erythrocytes. Here, we subjected rats to a collagenase-induced striatal ICH followed by whole-body TH (∼33℃ for 11-72 h) or normothermia. We used X-ray fluorescence imaging to spatially quantify total and peri-hematoma iron three days post-injury. At three and seven days, we measured non-heme iron levels. Finally, hematoma volume was quantified on one, three, and seven days. In the injured hemisphere, total iron levels were elevated ( p < 0.001) with iron increasing in the peri-hematoma region ( p = 0.007). Non-heme iron increased from three to seven days (p < 0.001). TH had no effect on any measure of iron ( p ≥ 0.479). At one and three days, TH did not affect hematoma volume ( p ≥ 0.264); however, at seven days there was a four-fold increase in hematoma volume in 40% of treated animals ( p = 0.032). Thus, even when TH does not interfere with initial increases in total and non-heme iron or its containment, TH can cause re-bleeding post-treatment. This serious complication could partly account for the intermittent protection previously observed. This also raises serious concerns for clinical usage of TH for ICH.