Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Stroke. 2012 Dec 18;44(1):205–212. doi: 10.1161/STROKEAHA.112.673954

Safety and Efficacy Evaluation of Carnosine, An Endogenous Neuroprotective Agent for Ischemic Stroke

Ok-Nam Bae 1,2, Kelsey Serfozo 1, Seung-Hoon Baek 3, Ki Yong Lee 1, Anne Dorrance 4, Wilson Rumbeiha 5, Scott D Fitzgerald 6, Muhammad U Farooq 1, Bharath Naravelta 1, Archit Bhatt 1, Arshad Majid 1,7
PMCID: PMC3678096  NIHMSID: NIHMS427679  PMID: 23250994

Abstract

Background and Purpose

An urgent need exists to develop therapies for stroke which have high efficacy, long therapeutic time windows and acceptable toxicity. We undertook preclinical investigations of a novel therapeutic approach involving supplementation with carnosine, an endogenous pleiotropic dipeptide.

Methods

Efficacy and safety of carnosine treatment was evaluated in rat models of permanent or transient middle cerebral artery occlusion. Mechanistic studies used primary neuronal/astrocytic cultures and ex vivo brain homogenates.

Results

Intravenous treatment with carnosine exhibited robust cerebroprotection in a dose-dependent manner, with long clinically-relevant therapeutic time windows of 6 h and 9 h in transient and permanent models, respectively. Histological outcomes and functional improvements including motor and sensory deficits were sustained at 14 d post-stroke onset. In safety and tolerability assessments, carnosine did not exhibit any evidence of adverse effects or toxicity. Moreover, histological evaluation of organs, complete blood count, coagulation tests and the serum chemistry did not reveal any abnormalities. In primary neuronal cell cultures and ex vivo brain homogenates, carnosine exhibited robust anti-excitotoxic, antioxidant, and mitochondria protecting activity.

Conclusion

In both permanent and transient ischemic models, carnosine treatment exhibited significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. Carnosine was well tolerated and exhibited no toxicity. Mechanistic data show that it influences multiple deleterious processes. Taken together, our data suggest that this endogenous pleiotropic dipeptide is a strong candidate for further development as a stroke treatment.

Keywords: carnosine, neuroprotection, ischemic stroke, efficacy, safety

Introduction

Despite extensive efforts to develop new treatments for ischemic stroke, many promising experimental drugs have failed in human clinical trials; many due to intolerable side effects, low efficacy, and short therapeutic time windows.1-4 Despite its benefits, the use of tissue plasminogen activator (tPA), which is the only approved acute drug therapy for ischemic stroke, is limited by the short therapeutic time window and the risk of hemorrhage.5,6 Therefore, an urgent need exists for safe and effective drugs.

Since tissue damage after stroke involves multiple deleterious mechanisms, it is desirable that novel therapeutic drugs favorably influence multiple molecular pathways that contribute to tissue damage.7,8 Carnosine is an endogenous dipeptide composed of alanine and histidine, and is expressed in many tissues of the body including the central nervous system.9 Carnosine exhibits pleiotropic biological activities such as antioxidant, cytosolic buffering, heavy metal chelating and anti-excitotoxic activity.10,11 Due to these beneficial and diverse activities, carnosine has been proposed as an attractive therapeutic candidate for ischemic stroke damage.

Carnosine reduces neurological impairment, decreases mortality, and improves functional outcome after global ischemia in gerbils and rats.12,13 In focal ischemia, we have previously shown that intraperitoneally administrated carnosine reduced brain damage in mouse ischemia models induced by permanent middle cerebral artery occlusion (pMCAO).14 We also showed that carnosine is the most effective among several carnosine analogues in reducing pMCAO-induced infarction volumes.15 Nevertheless, more thorough preclinical evaluation is needed for carnosine to meet the Stroke Therapeutic Academic Industry Roundtable (STAIR) guidelines and before clinical testing can take place.16

In the present study, we determined: 1) Short- and long-term neuroprotective efficacy of intravenous carnosine in rats using focal ischemia models (permanent and transient); 2) the therapeutic time window; 3) the safety and tolerability of carnosine; 4) the effect of carnosine on the thrombolytic activity of tPA; and 5) the influence of carnosine on several specific deleterious ischemia-induced mechanisms. Taken together, our findings provide strong support for the development of carnosine as a therapeutic agent for stroke.

Materials and Methods

More details are provided in the online data supplement.

Animals

Adult male Sprague-Dawley rats (250 to 300 g; Harlan) were used after approval from Institutional Animal Care and Use Committee at Michigan State University.

Blinding and Randomization

Treatment groups were allocated in a randomized fashion. Investigators were blind to the allocation.

Carnosine treatments

Carnosine (Sigma) was dissolved in saline and administered intravenously.

Permanent or transient middle cerebral artery occlusion (MCAO)

Permanent and transient focal cerebral ischemia was induced by placing and advancing a silicone-coated intra-luminal filament (Doccol Co.) in the carotid artery to occlude the middle cerebral artery. The filament was left in place for the permanent ischemia model. For the transient model, reperfusion was produced by withdrawal of the monofilament 3 hrs after occlusion. In experiments measuring the therapeutic time window, the filaments were removed 6 hr- or 9 hr after onset of ischemia.

Calculation of infarct volume by TTC staining

At 24 hr after onset of ischemia, rats were euthanized by isoflurane overdose, decapitated, and the brains were rapidly removed. The infarct volume was determined with correction for edema using triphenyltetrazolium chloride (TTC)-stained brain slices.

Assessment of neurological function

Neurological deficit was evaluated by an 18- point-based scale,17 adhesive tape removal testing, and accelerated rotarod testing.18

Quantification of brain damage with Nissl staining

The serial coronal sections (40 μm) were cut from the frozen brains and stained with cresyl violet. The infarct volume was estimated as the product of the sum of the lesion areas and the distance between sections.

Animal handling for safety assessment

Rats were allowed to survive for 14 d after carnosine treatments for safety assessment.

Histopathological evaluation of organ toxicity

Histopathological evaluation was performed on heart, lung, liver, kidney, brain, and bone marrow from four randomly selected animals from the group of saline or carnosine using Hematoxylin and Eosin staining.

Assessment of complete blood count/ coagulation and serum chemistry

Four randomly chosen rats from saline- or carnosine-treated group underwent the tests for complete blood count/coagulation and serum chemistry profiles at 14 days after carnosine administration.

Measurement of clot lysis

The effect of carnosine on the fibrinolytic activity of tPA was examined using spectrophotometry.

Primary neurons/astrocytes culture and determination of cytotoxicity

Primary cortical neuronal and astrocytic cultures were established as previously described,19 and used for experiments on days in vitro (DIV) 7-11 and DIV 14, respectively.

Measurement of reactive oxygen species (ROS) or mitochondrial membrane potential transition

The intracellular ROS levels or the mitochondrial membrane potential transition were evaluated in fluorescence microplate reader using dichlorofluorescein diacetate, acetyl ester (H2DCFDA) or JC-1, respectively.

Brain mitochondrial isolation and mitochondrial respiratory activity measurement

Brain mitochondria was isolated from rats after pMCAO, and the respiratory activity was measured using respiratory control ratio using a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK).20

Sample Size Estimates

The number of rats to be used per group was determined using a series of power calculations using commercially available software (Janet D. Elashoff, nQuery Advisor Version 2.0, Los Angeles, CA).

Statistics

Statistical analysis was performed using SPSS software (Chicago, IL) as described in online supplement. In all cases, a p value of <0.05 was considered significant.

Results

Improvement of histological and functional outcomes in rat transient focal ischemia

To examine the neuroprotective effect of carnosine, focal ischemic stroke was induced in rats using the intraluminal monofilament technique. No significant differences among the experimental groups were detected in physiological variables of body weight, rectal temperature, and cerebral blood flow before and after ischemia (Supplemental Table S1). Blockade and subsequent restoration of cerebral blood flow (CBF) was confirmed by laser Doppler. Carnosine did not induce any significant change in CBF (Fig. 1A).

Figure 1. Effects of carnosine on infarct size and neurological function in rats at 24 h after 3 h tMCAO.

Figure 1

Open in a new tab

A. Cerebral blood flow (CBF) was monitored during the surgical procedure. B. Effect of carnosine on neurological deficits. C. Left, representative TTC-stained sections; Right, quantification of the infarct volume. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=20, B:n=14-15, C:n=14-15. All values are means ± SEM and analyzed by ANOVA tests.

Carnosine was administered intravenously at 3 h after ischemia, and the monofilament was removed to allow reperfusion. Along with the functional improvement (Fig. 1B), treatment with carnosine (500 to 2000 mg/kg) significantly decreased brain damage in a dose-dependent manner (Fig. 1C). Carnosine treatment significantly decreased infarct volume by 41.9% (p=0.004) and 49.1% (p=0.002) at 1000 mg/kg and 2000 mg/kg dose, respectively.

Therapeutic time window

To determine the therapeutic time window, a single dose of carnosine was administered intravenously at increasing time intervals after ischemic stroke. Protective effect of carnosine was significant up to 6 h in the transient model (Fig. 2A), showing decrease of infarct volume by 41.9% (p=0.004), 39.4% (p=0.024) and 13.4% (p=0.882) for 3 h, 6 h and 9 h, respectively. The protective effect of carnosine was greater in the permanent ischemic model, where the time window was found to be 9 h (Fig. 2B). Carnosine reduced brain infarct by 57.1% (p<0.001), 41.2% (p=0.006), 30.7% (p=0.011) and 8.5% (p=0.500) for 3 h, 6 h, 9 h and 12 h post treatment following pMCAO, respectively.

Figure 2. Therapeutic time window of carnosine neuroprotection against ischemic stroke in rats.

Figure 2

Open in a new tab

Carnosine treatment was initiated at indicated time in transient- (A) or permanent-(B) ischemic rat models. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=7-15, B:13-15. All values are means ± SEM and analyzed by Student's t-test.

The influence of carnosine on mortality in permanent and transient ischemia

Stroke is associated with significant mortality. tPA improves disability but does not improve mortality. Our data show that carnosine is not only cerebroprotective, but it also improved mortality in both transient and permanent models. Mortality in the tMCAO model was 6.7% vs 0% after 3 h tMCAO, 13.3% vs 6.7% after 6 h tMCAO, and 65% vs 35% after 9 h tMCAO in saline-treated vs carnosine-treated rats, respectively. The decreased mortality in carnosine-treated rats was also observed in the permanent model over all the time-points tested (Supplemental Table S2).

Extended benefit of carnosine in rat transient focal ischemia

The neuroprotective effects of carnosine were apparent even 14 d after stroke onset. Rats received saline or carnosine (1000 mg/kg, i.v.) at 3 h after ischemia, and reperfusion was initiated immediately after administration of carnosine. At 14 d post-ischemia, histological brain damage was evaluated by Nissl staining. The brain sections from saline-treated rats exhibited a consistent necrotic lesion both in cortical and subcortical regions of ipsilateral hemisphere (Fig. 3A left). Infarct volumes were significantly decreased by carnosine treatment by 30.5% (Fig. 3A right; p=0.045).

Figure 3. Protective effects of carnosine in rats with 3 h-tMCAO through 14 d survival duration.

Figure 3

Open in a new tab

A. The distribution of infarct (left) and infarct volume (right) at 14 d after ischemia are shown. B to D. Functional outcome was evaluated by adhesive tape removal test (B), rotarod test (C), and neurological scoring (D). Sham rats underwent the same surgical procedure apart from MCAO. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=17-18, B:n=13-14, C:n=17, D=17-18 (Sham animal;n=5 for B to D). All values are means ± SEM and analyzed by Student's t-test.

Functional outcomes

We also examined whether treatment with carnosine influenced functional outcome. The adhesive tape removal test and the accelerated rotarod tests were used to assess responses/asymmetries and motor coordination/balance, respectively. Rats treated with carnosine showed a significant improvement in the adhesive tape removal test: 38.2% (p=0.031) at 7 d and by 44.9% (p=0.029) at 14 d after ischemia (Fig. 3B). Significant differences between saline- and carnosine-treated rats were also observed at 1 and 3 d after ischemic stroke using the rotarod test (Fig. 3C); 54.1% (p=0.006) and 71.8% (p=0.018), respectively. Differences were not statistically significant for the rotarod test at 7 d and 14d. Similarly, improvement in neurological scores was significant at all-time points observed through the14 d survival periods (Fig. 3D; p<0.01 for all time-points).

Assessment of safety and tolerability of carnosine

Next we examined the safety and tolerability of carnosine in rats. Based on the FDA guidelines on preclinical acute toxicity studies, daily assessments for systemic signs of toxicity were performed. Body weight, food consumption, activity, and mortality were evaluated for 14 d after single intravenous carnosine treatment (100, 500, 1000 and 2000 mg/kg). No significant differences were found between control (saline-treated) and carnosine-treated groups both in body weight change (Fig. 4A) and the amount of food consumption (Fig. 4B). No rats died in the control group or carnosine-treated groups.

Figure 4. Evaluation of safety and tolerability of carnosine after intravenous administration.

Figure 4

Open in a new tab

A and B, After single intravenous administration of saline or carnosine to rats, body weight (A) and food consumption (B) were monitored for 14 d. C, Histopathological evaluation of toxicity in various organs was performed using HE staining. A: n=7, B: n=7, C: n=4. All values are means ± SEM and analyzed by Student's t-test. Representative photos are shown.

To examine organ-specific toxicity, histopathological evaluations were performed on bone marrow, cerebellum, cerebrum, brain stem, hippocampus, heart, lung, liver, and kidney in randomly selected animals. Carnosine (2000 mg/kg) did not induce signs of toxicity in any of the examined organs (Fig. 4C).

Effects of carnosine on coagulation, complete blood count and serum chemistry

The effect of carnosine on coagulation, complete blood count (CBC), and serum chemistry were also examined. Fourteen days after treatment with saline or carnosine (2000 mg/kg), rats were sacrificed and blood was collected for analysis. No abnormalities were noted in any of the blood variables examined (Supplemental Table S3-S5).

Effect of carnosine on fibrinolytic activity of tPA

Next, we determined whether carnosine had any effect on clot lysis by tPA, since co-administration of tPA and carnosine may occur in a future clinical trial. Clots were generated from healthy volunteers. Exogenously added tPA significantly increased clot lysis, and plasminogen activator inhibitor (PAI) significantly attenuated clot lysis by t-PA, demonstrating the validity of the ex vivo assay. Carnosine itself had no apparent clot lysis activity (Fig. 5A). Significant clot lysis was obtained with tPA, achieving 50.7 ± 0.8 % total clot lysis after 120 min. Co-treatment with carnosine (10, 20, and 30 μg/mL) did not affect the thrombolytic activity of tPA (Fig. 5B).

Figure 5. The effect of carnosine on clot lysis by tPA.

Figure 5

Open in a new tab

The amount of clot lysis was obtained after addition of tPA or carnosine (CAR) using spectrophotometry. A, Control: No treatment, tPA: tissue plasminogen activator (8.3 μg/mL), PAI: plasminogen activator inhibitor (8.3 μg/mL), CAR: carnosine (30 μg/mL) B, The thrombolytic activity of tPA (8.3 μg/mL) was not affected by carnosine (10, 20, and 30 μg/mL). A: n=5, B: n=5. All values are means ± SEM.

Mechanisms underlying the protective effect of carnosine

To address how carnosine mediates cerebroprotective effects, we used primary cultures of cortical neurons and astrocytes, the major cell types impaired during ischemia.21 Carnosine reduced neuronal cell death induced by in vitro ischemic insults of oxygen-glucose deprivation (OGD) by 48.4%, or an excitotoxic stimulus of NMDA by 40.8% (Fig. 6A). Protective effects against OGD-induced injury were also observed in primary astrocytes (Fig. 6A). In both models, carnosine decreased reactive oxygen species generation, supporting its role as an antioxidant (Fig. 6B). Transition of mitochondrial membrane potential was protected by carnosine in cortical neurons as well as in astrocytes (Fig. 6C), suggesting that carnosine decreased mitochondrial damage. Moreover, in brain homogenates isolated after focal ischemia (pMCAO), mitochondrial respiratory damage in ipsilateral hemisphere22 was significantly recovered by carnosine treatment (1000 mg/kg, 6 h post-treatment)(Fig. 6D).

Figure 6. Underlying mechanisms for carnosine neuroprotection.

Figure 6

Open in a new tab

A to C, Primary cortical neurons or astrocytes were isolated from neonatal mice, and were exposed to ischemic stimulus of NMDA or oxygen-glucose deprivation (OGD). Cell death (A), generation of reactive oxygen species (B), and transition of mitochondrial membrane potential (C) were decreased by carnosine. *p<0.05 and **p<0.01 vs. ischemic stimulus. D. Effect of carnosine on respiratory control ratio was examined in ex vivo rat brain homogenates after pMCAO. *p<0.05 and **p<0.01. P/M, Pyruvate and malate; A, ADP; O, Oligomycine A; C, CCCP; S, Succinate. A: n=4, B: n=3,C: n=3, D: n=4. All values are means ± SEM and analyzed by ANOVA tests or by Student's t-test. Representative tracings of oxygen consumption are shown.

Discussion

To date, numerous neuroprotective agents have been effective in animal studies, but every agent has failed in clinical trials.23 Many reasons may account for this.1,24-26 Previous therapeutic strategies have targeted single pathways but stroke involves many different deleterious processes that eventually lead to cellular injury and cell death. Another point of concern has been the poor quality of animal studies with inadequate randomization, blinding, and appropriate statistical power. Many studies only evaluated acute histological endpoints whereas clinically stroke recovery is determined by functional capacity at delayed time-points. Consequently, guidelines by representatives from academia and industry (STAIR) have developed to improve the quality of pre-clinical studies.16,27 While this study follows STAIR recommendations, additional studies of carnosine in females, older animals, and animals with co-morbidities are still needed to more fully satisfy the recommendations.

The primary goal of this study was to determine preclinical efficacy, therapeutic time window, and safety of carnosine as a cerebroprotective therapy in stroke. We chose carnosine, an endogenous dipeptide, because of its beneficial pleiotropic effects on deleterious mechanisms that contribute to cell death during ischemia.7 We used intravenous dosing because in a future clinical trial carnosine would be administered intravenously. Our data show that carnosine is highly efficacious in protecting against brain damage when administered intravenously, and is safe and well tolerated at doses up to 2000 mg/kg in rats. Importantly, we demonstrate that carnosine is protective against both transient and permanent focal ischemia models. Testing in both models is important because, although permanent occlusion is more common in human patients, recanalization rates of about 30% have been reported. Many previous neuroprotective candidates only reported efficacy in one model.28-30

Another remarkable finding of our study is the wide and clinically useful therapeutic time window. A critical issue in stroke treatment is that many patients arrive in hospital several hours after their stroke onset. We observed significant protection even when carnosine therapy was initiated 6 h after the onset of tMCAO or 9 h after pMCAO (Fig. 2). The benefits of tPA have been limited primarily due to its narrow therapeutic time window of 4.5 h.31,32 Many agents that have failed in clinical testing had short preclinical therapeutic time windows but were tested clinically with longer inclusion time windows. This broad window of cerebroprotective efficacy demonstrated in this study makes carnosine an attractive therapeutic candidate.

In most preclinical studies of cerebroprotective agents, efficacy was evaluated by reductions in histologically-determined infarction volumes. However, cerebroprotective efficacy is measured by neurological function in clinical trials. Infarction volumes correlate poorly with functional outcome because small lesions in critical brain areas can result in major functional deficits, whereas large lesions in silent areas can cause little detectable dysfunction.33,34 We demonstrated that carnosine showed protective effects both histologically and functionally. We employed several functional tests as each functional test represents a specific damage of somatosensory, motor-ambulatory, and fine motor/tactile function. Carnosine improved deficits in all functional tests (Fig. 3C, D, and E), and these protective effects persisted through the 14 d-survival period.

Because several neuroprotective candidates were withdrawn from clinical testing due to their adverse effects, preclinical assessments of the safety and tolerability of a putative neuroprotective agent represent a critical translational step in moving a therapeutic from animals to humans. We observed the effect of carnosine on complete blood count (CBC), serum biochemistry, and coagulation. In addition, histopathological evaluations of several organs were performed to evaluate organ-specific adverse effects; however, in all analyses, no adverse effects were seen (Supplemental Table S2-S4 and Fig. 4). Moreover, we tested the effect of carnsoine on the ability of tPA to thrombolyse clot since carnosine may be co-administered with tPA in a future trial, and found that carnosine did not influence the thrombolytic function of tPA (Fig. 5). Future studies are planned that will test carnosine with tPA in a focal ischemia model. These studies will also determine the influence of carnosine on edema formation and hemorrhagic transformation of infarction.

A significant strength of carnosine is its beneficial effects against brain damage resulting from ischemic stroke is likely to be mediated through multiple mechanisms. The interruption of cerebral perfusion during stroke initiates a cascade of multiple detrimental events leading to cell death.4,35 These detrimental events include secondary inflammation, enhanced matrix metalloproteinase activity, excitotoxicity, apoptosis, free radical injury, and microglial activation. Previous neuroprotection strategies have focused on targeting single pathways. Recent studies have suggested that an ideal cerebroprotectant should favorably influence multiple pathways.7,8 Carnosine is reported to exhibit anti-oxidant, pH buffering, heavy metal chelating, anti-excitotoxic, and vasodilating effects in many cell types including neurons,36-39 as well as under various disease states.40-42 Carnosine may enhance neurogenesis which may also contribute to recovery after stroke.43 Here we demonstrated that carnosine reduced neuronal and astrocytic cell death against ischemia-like insults such as OGD and NMDA (Fig. 6A). Consistent with previous reports, carnosine showed anti-oxidant and mitochondrial-protecting activities in neuronal and astroglial cells. Of note, we documented that carnosine treatment at 6 h after pMCAO rescued mitochondrial respiratory function, which is critical for cell survival during ischemic stroke.20,22 Future studies will evaluate functional improvement at longer intervals after ischemia onset. Moreover, studies are planned in aged animals and animals that have co-morbidities like hypertension and diabetes. Although we and others have shown that carnosine can penetrate the blood-brain barrier of rodents,15,44,45 detailed data on brain pharmacokinetics have not been generated.

In summary, using both histological and functional outcome metrics, we documented that intravenous carnosine confers enduring brain protection against focal ischemic stroke. Carnosine was safe and well tolerated at efficacious doses and has a wide, clinically-useful therapeutic time window. Our findings support the therapeutic potential of carnosine for ischemic stroke, and will be used to plan further preclinical and clinical testing to fully satisfy STAIR before clinical testing.

Supplementary Material

1

Acknowledgments

Source of Funding: This study was supported by the AHA grant to Arshad Majid MD and by Basic Science Research Program through the National Research Foundation of Korea (NRF) to Ok-Nam Bae (2012R1A1A3013240).

Footnotes

Conflict(s) of Interest/Disclosure(s): None

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Fisher M. New approaches to neuroprotective drug development. Stroke. 2011;42:S24–27. doi: 10.1161/STROKEAHA.110.592394. [DOI] [PubMed] [Google Scholar]
  • 2.Hess DC. NSY-059: A hopeful sign in the treatment of stroke. Stroke. 2006;37:2649–2650. doi: 10.1161/01.STR.0000240511.22775.a7. [DOI] [PubMed] [Google Scholar]
  • 3.Kennedy J, Buchan AM. C-EPO: ready for prime-time preconditioning? Cerebrovasc Dis. 2005;19:272–273. doi: 10.1159/000084140. [DOI] [PubMed] [Google Scholar]
  • 4.Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci. 2003;4:399–415. doi: 10.1038/nrn1106. [DOI] [PubMed] [Google Scholar]
  • 5.Ginsberg MD. Current status of neuroprotection for cerebral ischemia: synoptic overview. Stroke. 2009;40:S111–114. doi: 10.1161/STROKEAHA.108.528877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Wardlaw JM, Murray V, Berge E, del Zoppo G, Sandercock P, Lindley RL, et al. Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet. 2012;379:2364–2372. doi: 10.1016/S0140-6736(12)60738-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Auriel E, Bornstein NM. Neuroprotection in acute ischemic stroke--current status. J Cell Mol Med. 2010;14:2200–2202. doi: 10.1111/j.1582-4934.2010.01135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Green AR, Shuaib A. Therapeutic strategies for the treatment of stroke. Drug Discov Today. 2006;11:681–693. doi: 10.1016/j.drudis.2006.06.001. [DOI] [PubMed] [Google Scholar]
  • 9.De Marchis S, Modena C, Peretto P, Migheli A, Margolis FL, Fasolo A. Carnosine-related dipeptides in neurons and glia. Biochemistry (Mosc) 2000;7:969–980. [PubMed] [Google Scholar]
  • 10.Guiotto A, Calderan A, Ruzza P, Borin G. Carnosine and carnosine-related antioxidants: a review. Curr Med Chem. 2005;12:2293–2315. doi: 10.2174/0929867054864796. [DOI] [PubMed] [Google Scholar]
  • 11.Hipkiss AR. Carnosine and its possible roles in nutrition and health. Adv Food Nutr Res. 2009;57:87–154. doi: 10.1016/S1043-4526(09)57003-9. [DOI] [PubMed] [Google Scholar]
  • 12.Dobrota D, Fedorova T, Stvolinsky S, Babusikova E, Likavcanova K, Drgova A, et al. Carnosine protects the brain of rats and Mongolian gerbils against ischemic injury: after-stroke-effect. Neurochem Res. 2005;30:1283–1288. doi: 10.1007/s11064-005-8799-7. [DOI] [PubMed] [Google Scholar]
  • 13.Gallant S, Kukley M, Stvolinsky S, Bulygina E, Boldyrev A. Effect of carnosine on rats under experimental brain ischemia. Tohoku J Exp Med. 2000;191:85–99. doi: 10.1620/tjem.191.85. [DOI] [PubMed] [Google Scholar]
  • 14.Rajanikant GK, Zemke D, Senut MC, Frenkel MB, Chen AF, Gupta R, et al. Carnosine is neuroprotective against permanent focal cerebral ischemia in mice. Stroke. 2007;38:3023–3031. doi: 10.1161/STROKEAHA.107.488502. [DOI] [PubMed] [Google Scholar]
  • 15.Min J, Senut MC, Rajanikant K, Greenberg E, Bandagi R, Zemke D, et al. Differential neuroprotective effects of carnosine, anserine, and N-acetyl carnosine against permanent focal ischemia. J Neurosci Res. 2008;86:2984–2991. doi: 10.1002/jnr.21744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40:2244–2250. doi: 10.1161/STROKEAHA.108.541128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Garcia JH, Wagner S, Liu KF, Hu XJ. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke. 1995;26:627–634. doi: 10.1161/01.str.26.4.627. discussion 635. [DOI] [PubMed] [Google Scholar]
  • 18.Wang X, Liu J, Zhu H, Tejuma W, Tsuju K, Murata Y, et al. Effects of neuroglobin overexpression on acute brain injury and long-term outcomes after focal cerebral ischemia. Stroke. 2008;39:1869–1874. doi: 10.1161/STROKEAHA.107.506022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bae ON, Rajanikant K, Min J, Smith J, Baek SH, Serfozo K, et al. Lck activation mediates neuroprotection during ischemic preconditioning. J Neurosci. 2012;32:7278–7286. doi: 10.1523/JNEUROSCI.6273-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Loh KP, Tan BK, Liu XH, Wei BG, Zhu YZ. Leonurine protects middle cerebral artery occluded rats through antioxidant effect and regulation of mitochondrial function. Stroke. 2010;41:2661–2668. doi: 10.1161/STROKEAHA.110.589895. [DOI] [PubMed] [Google Scholar]
  • 21.Rossi DJ, Brady JD, Mohr C. Astrocyte metabolism and signaling during brain ischemia. Nat Neurosci. 2007;10:1377–1386. doi: 10.1038/nn2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Brown MR, Sullivan PG, Geddes JW. Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria. J Biol Chem. 2006;281:11658–11668. doi: 10.1074/jbc.M510303200. [DOI] [PubMed] [Google Scholar]
  • 23.Green AR. Pharmacological approaches to acute ischaemic stroke: reperfusion certainly, neuroprotection possibly. Br J Pharmacol. 2008;153(1):S325–338. doi: 10.1038/sj.bjp.0707594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.O'Collins VE, Macleod MR, Cox SF, Van Raay L, Aleksoska E, Donnan GA, et al. Preclinical drug evaluation for combination therapy in acute stroke using systematic review, meta-analysis, and subsequent experimental testing. J Cereb Blood Flow Metab. 2011;31:962–975. doi: 10.1038/jcbfm.2010.184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Philip M, Benatar M, Fisher M, Savitz SI. Methodological quality of animal studies of neuroprotective agents currently in phase II/III acute ischemic stroke trials. Stroke. 2009;40:577–581. doi: 10.1161/STROKEAHA.108.524330. [DOI] [PubMed] [Google Scholar]
  • 26.Rogalewski A, Schneider A, Ringelstein EB, Schabitz WR. Toward a multimodal neuroprotective treatment of stroke. Stroke. 2006;37:1129–1136. doi: 10.1161/01.STR.0000209330.73175.34. [DOI] [PubMed] [Google Scholar]
  • 27.Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999;30:2752–2758. doi: 10.1161/01.str.30.12.2752. [DOI] [PubMed] [Google Scholar]
  • 28.Britton P, Lu XC, Laskosky MS, Tortella FC. Dextromethorphan protects against cerebral injury following transient, but not permanent, focal ischemia in rats. Life Sci. 1997;60:1729–1740. doi: 10.1016/s0024-3205(97)00132-x. [DOI] [PubMed] [Google Scholar]
  • 29.Morikawa E, Zhang SM, Seko Y, Toyoda T, Kirino T. Treatment of focal cerebral ischemia with synthetic oligopeptide corresponding to lectin domain of selectin. Stroke. 1996;27:951–955. doi: 10.1161/01.str.27.5.951. discussion 956. [DOI] [PubMed] [Google Scholar]
  • 30.Xue D, Slivka A, Buchan AM. Tirilazad reduces cortical infarction after transient but not permanent focal cerebral ischemia in rats. Stroke. 1992;23:894–899. doi: 10.1161/01.str.23.6.894. [DOI] [PubMed] [Google Scholar]
  • 31.Davis SM, Donnan GA. 4.5 hours: the new time window for tissue plasminogen activator in stroke. Stroke. 2009(40):2266–2267. doi: 10.1161/STROKEAHA.108.544171. [DOI] [PubMed] [Google Scholar]
  • 32.Lansberg MG, Bluhmki E, Thijs VN. Efficacy and safety of tissue plasminogen activator 3 to 4.5 hours after acute ischemic stroke: a metaanalysis. Stroke. 2009;40:2438–2441. doi: 10.1161/STROKEAHA.109.552547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Allred RP, Jones TA. Maladaptive effects of learning with the less-affected forelimb after focal cortical infarcts in rats. Exp Neurol. 2008;210:172–181. doi: 10.1016/j.expneurol.2007.10.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Schaar KL, Brenneman MM, Savitz SI. Functional assessments in the rodent stroke model. ExpTransl Stroke Med. 2010;2:13. doi: 10.1186/2040-7378-2-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang X, Lee SR, Arai K, Lee SR, Tsuju K, Rebeck GW, et al. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003;9:1313–1317. doi: 10.1038/nm926. [DOI] [PubMed] [Google Scholar]
  • 36.Calabrese V, Colombrita C, Guagliano E, Sapienza M, Ravagna A, Cardile V, et al. Protective effect of carnosine during nitrosative stress in astroglial cell cultures. Neurochem Res. 2005;30:797–807. doi: 10.1007/s11064-005-6874-8. [DOI] [PubMed] [Google Scholar]
  • 37.Nicoletti VG, Santoro AM, Grasso G, Vagliasindi LI, Giuffrida ML, Cuppari C, et al. Carnosine interaction with nitric oxide and astroglial cell protection. J Neurosci Res. 2007;85:2239–2245. doi: 10.1002/jnr.21365. [DOI] [PubMed] [Google Scholar]
  • 38.Shen Y, Hu WW, Fan YY, Dai HB, Fu QL, Wei EQ, et al. Carnosine protects against NMDA-induced neurotoxicity in differentiated rat PC12 cells through carnosine-histidine-histamine pathway and H(1)/H(3) receptors. Biochem Pharmacol. 2007;73:709–717. doi: 10.1016/j.bcp.2006.11.007. [DOI] [PubMed] [Google Scholar]
  • 39.Trombley PQ, Horning MS, Blakemore LJ. Interactions between carnosine and zinc and copper: implications for neuromodulation and neuroprotection. Biochemistry (Mosc) 2000;65:807–816. [PubMed] [Google Scholar]
  • 40.Jin CL, Yang LX, Wu XH, Li Q, Ding MP, Fan YY, et al. Effects of carnosine on amygdaloid-kindled seizures in Sprague-Dawley rats. Neuroscience. 2005;135:939–947. doi: 10.1016/j.neuroscience.2005.06.066. [DOI] [PubMed] [Google Scholar]
  • 41.Lee YT, Hsu CC, Lin MH, Liu KS, Yin MC. Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation. Eur J Pharmacol. 2005;513:145–150. doi: 10.1016/j.ejphar.2005.02.010. [DOI] [PubMed] [Google Scholar]
  • 42.Shen Y, He P, Fan YY, Zhang JX, Yan HJ, Hu WW, et al. Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity. Free Radic Biol Med. 2010;48:727–735. doi: 10.1016/j.freeradbiomed.2009.12.021. [DOI] [PubMed] [Google Scholar]
  • 43.Perroteau I, Biffo S, Tolosano E, Tarozzo G, Bovolin P, Vaudry H, et al. In vitro study of olfactory receptor neurones expressing the dipeptide carnosine. Neuroreport. 1994;5:569–572. doi: 10.1097/00001756-199401000-00009. [DOI] [PubMed] [Google Scholar]
  • 44.Stvolinsky S, Kukley M, Dobrota D, Mezesova V, Boldyrev A. Carnosine protects rats under global ischemia. Brain Res Bull. 2000;53:445–448. doi: 10.1016/s0361-9230(00)00366-x. [DOI] [PubMed] [Google Scholar]
  • 45.Jin CL, Yang LX, Wu XH, Li Q, Ding MP, Fan YY, et al. Effects of carnosine on amygdaloid-kindled seizures in Sprague-Dawley rats. Neuroscience. 2005;135:939–947. doi: 10.1016/j.neuroscience.2005.06.066. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS427679-supplement-1.pdf (155.7KB, pdf)

RESOURCES