The deleterious effect of crossfostering in rat pups on hypoxic-ischemic injury tolerance and hypothermic neuroprotection

We study the effect of hypothermia (HT) following hypoxic-ischemic (HI) brain injury in postnatal day 7 (P7) rats. In 2015, new European Union animal transport regulations prompted a change in practice at the breeding facility, which henceforth crossfostered P3 litters to P8 older lactating dam prior to transportation. It is generally assumed that crossfostering does not significantly affect the experimental results. The aim of this study was to examine whether crossfostering affects our model consistency by modifying injury susceptibility and hypothermic neuroprotection. We analysed 219 pups (56 litters) from 11 experiments conducted between 2013 and 2015: 73 non-crossfostered and 146 crossfostered pups. At P7, all pups underwent unilateral common carotid artery ligation followed by 50min of hypoxia (8% O2, 36°C). Immediately after this mild insult, the pups were randomised to post-insult normothermia (NT) or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of treatment group. Hypothermic neuroprotection was statistically significant, but with a smaller and less consistent effect in crossfostered pups (relative neuroprotection 16% vs. 31% in non-crossfostered). These results demonstrate hypothermic neuroprotection following a mild HI insult. A representative subset of 41 animals were also assessed for evidence of microglial reactivity, however no detectable difference in microglial reactivity was observed between any of the groups. In conclusion, crossfostering alters outcomes in our established model through reduced insult tolerance and variable neuroprotection. Crossfostering as a common breeding practice is a largely unexplored variable in animal research that may result in invalid research conclusions if inadequately adjusted for by larger group sizes. As a result, crossfostering is likely to be inconsistent with the principles of replacement, reduction, and refinement.

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Artemin is Upregulated by TrkB agonist and Protects the Immature Retina against Hypoxic-ischemic Injury by Suppressing Neuroinflammation and Astrogliosis

10.21203/rs.3.rs-115996/v1 ◽

2020 ◽

Author(s):

Hsiu-Mei Huang ◽

Chao-Ching Huang ◽

Linda Yi-Chieh Poon ◽

Ying-Chao Chang

Keyword(s):

Protective Effect ◽

Target Genes ◽

Neuroprotective Effect ◽

Neutralizing Antibody ◽

Developed Countries ◽

Artery Ligation ◽

Neuroprotective Effects ◽

Rat Pups ◽

Carotid Artery Ligation

Abstract Background: Hypoxic-ischemia (HI) is a major cause of acquired visual impairment in children of developed countries. Previous studies have shown that systemic administration of 7,8-dihydroxyﬂavone (DHF), a selective tropomyosin receptor kinase B (TrkB) agonist, provided long-term neuroprotection against HI injury in immature retina. However, the target genes and the mechanisms of the neuroprotective effects of TrkB signaling are not known. Methods: HI retinal injury was induced by unilateral common carotid artery ligation followed by 8% oxygen for 2 hrs at P7 rat pups. DHF was administered intraperitoneally at 2 hrs before and 18 hrs after HI injury. Polymerase chain reaction (PCR) array was used to identify genes upregulated after DHF treatment, then confirmed with quantitative real-time reverse transcriptase PCR and Western blot. Effects of the downstream mediator of DHF were assessed by intravitreal injection of neutralizing antibody at 4 hrs after DHF administration (24 hrs after HI). Meanwhile, the target protein was injected into the vitreous at 24 hrs after HI to validate its protective effect when exogenously supplemented. The outcomes were assessed by electroretinography and by histopathological sections of the rat retina. Results: Systemic DHF treatment after HI significantly increased the expression of artemin (ARTN) gene and protein at P8 and P10, respectively. The neuroprotective effects of DHF were inhibited after the blockade of ARTN protein with an increase in neuroinflammation and astrogliosis. ARTN treatment showed long-term protection against HI injury at both the histopathological and functional levels. The neuroprotective effects of ARTN were related to a decrease in microglial activation at P17, and attenuation of astrogliosis at P29. ARTN enhances phosphorylation of RET, ERK, and JNK, but not AKT or p38 in the immature retina. Conclusions: Neuroprotective effect of TrkB agonist is partially exerted through a mechanism that involves ARTN because the protective effect is ameliorated by ARTN sequestration. ARTN treatment after HI injury protects the immature retina by attenuating the late neuroinflammation and astrogliosis in the immature retina via ARTN/RET/JNK/ERK signaling pathway. ARTN can be a strategy to provide long-term protection in immature retina against HI injury.

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Carbon Dioxide Protects the Perinatal Brain From Hypoxic-Ischemic Damage: An Experimental Study in the Immature Rat

PEDIATRICS ◽

10.1542/peds.95.6.868 ◽

1995 ◽

Vol 95 (6) ◽

pp. 868-874 ◽

Cited By ~ 1

Author(s):

Robert C. Vannucci ◽

Javad Towfighi ◽

Daniel F. Heitjan ◽

Robert M. Brucklacher

Keyword(s):

Carbon Dioxide ◽

Carotid Artery ◽

Brain Damage ◽

Artery Ligation ◽

Immature Rat ◽

Rat Pups ◽

Carotid Artery Ligation ◽

Respiratory Management ◽

Systemic Hypoxia ◽

Hypoxia Ischemia

Background and Objective. Clinical investigations suggest that premature infants who require mechanical ventilation from respiratory distress syndrome are at increased risk for periventricular leukomalacia if hypocapnia occurs during respiratory management. The question remains as to the contribution of hypocapnia to hypoxic-ischemic brain damage and whether or not hypercapnia is neuroprotective. Methods. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed thereafter by exposure to systemic hypoxia with 8% oxygen (O2) combined with either 0, 3, 6, or 9% carbon dioxide (CO2) for 2.5 hours at 37°C. Survivors underwent neuropathologic examination at 30 days of postnatal age, and their brains were categorized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = atrophy with cystic cavitation <3 mm; 4 = cystic cavitation >3 mm of the cerebral hemisphere ipsilateral to the carotid artery ligation. The width of the ipsilateral hemisphere also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. Data were analyzed by linear models. Results. CO2 tensions averaged 26, 42, 54, and 71 mm Hg in the 0, 3, 6, and 9% CO2 exposed animals, respectively, during systemic hypoxia. Blood O2 tensions during hypoxia were not different among the four groups and averaged 34.7 mm Hg. Neuropathologic results showed that 30/38 (79%) rats exposed to 3% CO2 showed either no or mild brain damage compared with 13/33 (39%) controls (0% CO2). Cystic cavitation occurred in only four CO2 exposed rat pups compared with 14 controls (P = .001). At 6% CO2 exposure, all of 20 rat pups showed either no damage or mild atrophy compared with controls (P < .001); and at 9% CO2 exposure, 19/23 (83%) rat pups showed no or mild damage compared with controls (P < .001). The data also showed that the greatest reduction in brain damage occurred in immature rats exposed to 6% CO2 with slightly less protection at 9% CO2 (P = .012), the latter comparable with the severity of brain damage sustained by animals inhaling 3% CO2. Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. Conclusions. The results indicate that in an immature rat model normocapnic cerebral hypoxia-ischemia is associated with less severe brain damage than in hypocapnic hypoxia-ischemia and that mild hypercapnia is more protective than normocapnia. The findings in an experimental model merit further animal investigations as well as a clinical reappraisal of the ventilatory management of sick newborn human infants.

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Cerebrovascular Adaptation after Unilateral Carotid Artery Ligation in the Rat: Preservation of Blood Flow and ATP during Forebrain Ischemia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199801000-00012 ◽

1998 ◽

Vol 18 (1) ◽

pp. 118-121 ◽

Cited By ~ 16

Author(s):

Guri Bronner ◽

Kendall Mitchell ◽

Frank A. Welsh

Keyword(s):

Blood Flow ◽

Carotid Artery ◽

Forebrain Ischemia ◽

Artery Ligation ◽

Ipsilateral Hemisphere ◽

Cortical Blood Flow ◽

Carotid Artery Ligation ◽

Atp Levels ◽

Male Wistar Rats

To investigate long-term adaptations after unilateral carotid artery ligation, the effect of forebrain ischemia on cerebral blood flow and ATP levels was determined at various times after ligation. Unilateral carotid artery ligation was performed in male Wistar rats 0, 3, or 7 days before forebrain ischemia. Laser-Doppler blood flow was monitored bilaterally over the parietal cortex and ATP was measured in the subadjacent cortex of both hemispheres at the end of a 10-minute episode of ischemia. In the 0-day group, forebrain ischemia reduced cortical blood flow to 12% ± 8% (mean ± SD) of preischemic values and lowered cortical ATP to 26% ± 35% of control levels in the ipsilateral hemisphere. Delaying the onset of forebrain ischemia for 3 days after carotid artery ligation significantly improved cortical blood flow (29% ± 12%, P < 0.05) and ATP levels (92% ± 11%, P < 0.05) in the ipsilateral hemisphere. Delaying forebrain ischemia for 7 days also significantly improved ipsilateral blood flow (36% ± 11%, P < 0.05) and ATP levels (81% ± 29%, P < 0.05) compared with the 0-day group. In the contralateral hemisphere, the reduction in blood flow and ATP levels was not significantly altered by delaying the onset of forebrain ischemia for 3 or 7 days. These results show that unilateral carotid artery ligation induces long-term vascular adaptations that improve the collateral circulation and preserve ATP levels during a subsequent episode of ischemia.

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Columnar Alterations of NADH Fluorescence during Hypoxia-Ischemia in Immature Rat Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1982.22 ◽

1982 ◽

Vol 2 (2) ◽

pp. 221-228 ◽

Cited By ~ 105

Author(s):

Frank A. Welsh ◽

Robert C. Vannucci ◽

James B. Brierley

Keyword(s):

Recovery Period ◽

High Energy ◽

High Energy Phosphates ◽

Contralateral Hemisphere ◽

Artery Ligation ◽

Ipsilateral Hemisphere ◽

Carotid Artery Ligation ◽

Systemic Hypoxia ◽

Nadh Fluorescence ◽

Hypoxia Ischemia

Cerebral hypoxia-ischemia was produced in 7-day postnatal rats by unilateral carotid artery ligation combined with systemic hypoxia (8% O2). Levels of high energy phosphates, which were only slightly altered in the contralateral hemisphere, were nearly depleted in the ipsilateral hemisphere during the 3-h hypoxic insult. With hypoxia of between 1 and 3 hours' duration, columnar alterations of cortical NADH fluorescence occurred in the same location and regional pattern as did histologic damage demonstrated previously (Rice et al., 1981). In regions exhibiting columns of NADH fluorescence, there was no evidence of a columnar reduction of high energy phosphates as levels of ATP and phosphocreatine were nearly zero. Recovery from 3 h of hypoxia was accompanied by partial and regionally heterogeneous restoration of ATP within the ipsilateral hemisphere. Columnar variations of NADH fluorescence were not detected in the recovery period; rather, regions with impaired restitution of high energy phosphates exhibited NADH fluorescence that was diminished diffusely compared to the contralateral hemisphere. The correlation between depressed NADH fluorescence and depleted ATP, present as cortical columns during hypoxia and as larger regions during recovery, suggests that decreased formation of NADH may be limiting the resynthesis of high energy phosphates.

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Temporal Changes of Regional Glucose Use, Blood Flow, and Microtubule-Associated Protein 2 Immunostaining after Hypoxia—Ischemia in the Immature Rat Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199802000-00014 ◽

1998 ◽

Vol 18 (2) ◽

pp. 222-228 ◽

Cited By ~ 49

Author(s):

Eric Gilland ◽

Elsa Bona ◽

Henrik Hagber

Keyword(s):

Secondary Phase ◽

Contralateral Side ◽

Energy Utilization ◽

Artery Ligation ◽

Early Reperfusion ◽

Immature Rat ◽

Ipsilateral Hemisphere ◽

Carotid Artery Ligation ◽

Hypoxia Ischemia ◽

Old Rats

In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia–ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.

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The Effect of Hyperglycemia on Cerebral Metabolism during Hypoxia-Ischemia in the Immature Rat

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-199609000-00028 ◽

1996 ◽

Vol 16 (5) ◽

pp. 1026-1033 ◽

Cited By ~ 58

Author(s):

Robert C. Vannucci ◽

Robert M. Brucklacher ◽

Susan J. Vannucci

Keyword(s):

Cerebral Metabolism ◽

Glycolytic Flux ◽

Artery Ligation ◽

Immature Rat ◽

Ischemic Brain ◽

Glucose Levels ◽

Rat Pups ◽

Carotid Artery Ligation ◽

Hypoxia Ischemia ◽

Blood Glucose Concentrations

Unlike adults, hyperglycemia with circulating glucose concentrations of 25–35 m M/L protects the immature brain from hypoxic–ischemic damage. To ascertain the effect of hyperglycemia on cerebral oxidative metabolism during the course of hypoxia–ischemia, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% O2 for 2 h at 37°C. Experimental animals received 0.2 cc s.c. 50% glucose at the onset of hypoxia–ischemia, and 0.15 cc 25% glucose 1 h later to maintain blood glucose concentrations at 20–25 m ML for 2 h. Control rat pups received equivalent concentrations or volumes of either mannitol or 1 N saline at the same intervals. The cerebral metabolic rate for glucose (CMRglc.) increased from 7.1 (control) to 20.2 μmol 100 g−1 min−1 in hyperglycemic rats during the first hour of hypoxia–ischemia, 79 and 35% greater than the rates for saline- and mannitol-injected animals at the same interval, respectively ( p < 0.01). Brain intracellular glucose concentrations were 5.2 and 3.0 m M/kg in the hyperglycemic rat pups at 1 and 2 h of hypoxia–ischemia, respectively; glucose levels were near negligible in mannitol- and saline-treated animals at the same intervals. Brain intracellular lactate concentrations averaged 13.4 and 23.3 m M/kg in hyperglycemic animals at 1 and 2 h of hypoxia–ischemia, respectively, more than twice the concentrations estimated for the saline- and mannitol-treated littermates. Phosphocreatine (PCr) and ATP decreased in all three experimental groups, but were preserved to the greatest extent in hyperglycemic animals. Results indicate that anaerobic glycolytic flux is increased to a greater extent in hyperglycemic immature rats than in normoglycemic littermates subjected to cerebral hypoxia–ischemia, and that the enhanced glycolysis leads to greater intracellular lactate accumulation. Despite cerebral lactosis, energy reserves were better preserved in hyperglycemic animals than in saline-treated controls, thus accounting for the greater resistance of hyperglycemic animals to hypoxic–ischemic brain damage.

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Artemin Is Upregulated by TrkB Agonist and Protects the Immature Retina Against Hypoxic-Ischemic Injury by Suppressing Neuroinflammation and Astrogliosis

Frontiers in Molecular Neuroscience ◽

10.3389/fnmol.2021.645000 ◽

2021 ◽

Vol 14 ◽

Author(s):

Hsiu-Mei Huang ◽

Chao-Ching Huang ◽

Linda Yi-Chieh Poon ◽

Ying-Chao Chang

Keyword(s):

Protective Effect ◽

Target Genes ◽

Neuroprotective Effect ◽

Neutralizing Antibody ◽

Developed Countries ◽

Artery Ligation ◽

Neuroprotective Effects ◽

Rat Pups ◽

Carotid Artery Ligation

Hypoxic-ischemia (HI) is a major cause of acquired visual impairment in children from developed countries. Previous studies have shown that systemic administration of 7,8-dihydroxyavone (DHF), a selective tropomyosin receptor kinase B (TrkB) agonist, provides long-term neuroprotection against HI injury in an immature retina. However, the target genes and the mechanisms of the neuroprotective effects of TrkB signaling are not known. In the present study, we induced an HI retinal injury through unilateral common carotid artery ligation followed by 8% oxygen for 2 h in P7 rat pups. DHF was administered intraperitoneally 2 h before and 18 h after the HI injury. A polymerase chain reaction (PCR) array was used to identify the target genes upregulated after the DHF treatment, which was then confirmed with quantitative real-time reverse transcriptase PCR and a western blot. Effects of the downstream mediator of DHF were assessed using an intravitreal injection of neutralizing antibody 4 h after DHF administration (24 h after HI). Meanwhile, the target protein was injected into the vitreous 24 h after HI to validate its protective effect when exogenously supplemented. We found that systemic DHF treatment after HI significantly increased the expression of the artemin (ARTN) gene and protein at P8 and P10, respectively. The neuroprotective effects of DHF were inhibited after the ARTN protein blockade, with an increase in neuroinflammation and astrogliosis. ARTN treatment showed long-term protection against HI injury at both the histopathological and functional levels. The neuroprotective effects of ARTN were related to a decrease in microglial activation at P17 and attenuation of astrogliosis at P29. ARTN enhances phosphorylation of RET, ERK, and JNK, but not AKT or p38 in the immature retina. Altogether, these results suggest that the neuroprotective effect of a TrkB agonist is partially exerted through a mechanism that involves ARTN because the protective effect is ameliorated by ARTN sequestration. ARTN treatment after HI injury protects the immature retina by attenuating late neuroinflammation and astrogliosis in the immature retina relating to the ARTN/RET/JNK/ERK signaling pathway. ARTN may be a strategy by which to provide long-term protection in the immature retina against HI injury.

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Umbilical cord blood therapy modulates neonatal hypoxic ischemic brain injury in both females and males

Scientific Reports ◽

10.1038/s41598-021-95035-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tayla R. Penny ◽

Yen Pham ◽

Amy E. Sutherland ◽

Joohyung Lee ◽

Graham Jenkin ◽

...

Keyword(s):

Brain Injury ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Significant Risk ◽

Immunohistochemical Analysis ◽

Significant Risk Factor ◽

Tissue Loss ◽

Artery Ligation ◽

Rat Pups

AbstractPreclinical and clinical studies have shown that sex is a significant risk factor for perinatal morbidity and mortality, with males being more susceptible to neonatal hypoxic ischemic (HI) brain injury. No study has investigated sexual dimorphism in the efficacy of umbilical cord blood (UCB) cell therapy. HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received 3 doses of UCB cells (PND11, 13, 20) and underwent behavioural testing. On PND50, brains were collected for immunohistochemical analysis. Behavioural and neuropathological outcomes were assessed for sex differences. HI brain injury resulted in a significant decrease in brain weight and increase in tissue loss in females and males. Females and males also exhibited significant cell death, region-specific neuron loss and long-term behavioural deficits. Females had significantly smaller brains overall compared to males and males had significantly reduced neuron numbers in the cortex compared to females. UCB administration improved multiple aspects of neuropathology and functional outcomes in males and females. Females and males both exhibited injury following HI. This is the first preclinical evidence that UCB is an appropriate treatment for neonatal brain injury in both female and male neonates.

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Contribution of PDGFRα-positive cells in maintenance and injury responses in mouse large vessels

Scientific Reports ◽

10.1038/s41598-021-88126-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kenichi Kimura ◽

Karina Ramirez ◽

Tram Anh Vu Nguyen ◽

Yoshito Yamashiro ◽

Aiko Sada ◽

...

Keyword(s):

Growth Factor Receptor ◽

Pressure Overload ◽

Vascular Diseases ◽

Cell Types ◽

Lineage Tracing ◽

Neointima Formation ◽

Artery Ligation ◽

Carotid Artery Ligation ◽

Factor Receptor Alpha ◽

Adventitial Cells

AbstractThe maladaptive remodeling of vessel walls with neointima formation is a common feature of proliferative vascular diseases. It has been proposed that neointima formation is caused by the dedifferentiation of mature smooth muscle cells (SMCs). Recent evidence suggests that adventitial cells also participate in neointima formation; however, their cellular dynamics are not fully understood. In this study, we utilized a lineage tracing model of platelet-derived growth factor receptor alpha (PDGFRa) cells and examined cellular behavior during homeostasis and injury response. PDGFRa marked adventitial cells that were largely positive for Sca1 and a portion of medial SMCs, and both cell types were maintained for 2 years. Upon carotid artery ligation, PDGFRa-positive (+) cells were slowly recruited to the neointima and exhibited an immature SMC phenotype. In contrast, in a more severe wire denudation injury, PDGFRa+ cells were recruited to the neointima within 14 days and fully differentiated into SMCs. Under pressure overload induced by transverse aortic constriction, PDGFRa+ cells developed marked adventitial fibrosis. Taken together, our observations suggest that PDGFRa+ cells serve as a reservoir of adventitial cells and a subset of medial SMCs and underscore their context-dependent response to vascular injuries.

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Common carotid artery ligation at the proximal side before rupture in patients with ligation or occlusion of the external carotid artery at risk of carotid blowout syndrome

Japanese Journal of Clinical Oncology ◽

10.1093/jjco/hyz075 ◽

2019 ◽

Vol 49 (9) ◽

pp. 839-844

Author(s):

Fumihiko Matsumoto ◽

Satoko Matsumura ◽

Taisuke Mori ◽

Ayaka Mori ◽

Go Omura ◽

...

Keyword(s):

High Risk ◽

Carotid Artery ◽

Common Carotid Artery ◽

External Carotid Artery ◽

External Carotid ◽

Artery Ligation ◽

Bleeding Point ◽

Carotid Blowout Syndrome ◽

Proximal Side ◽

Carotid Artery Ligation

CCA ligation at the proximal side of the bleeding point under local anesthesia and before rupture is a treatment option for patients at high risk of CBS.

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