• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Stroke. Author manuscript; available in PMC Feb 1, 2011.
Published in final edited form as:
PMCID: PMC2822348
NIHMSID: NIHMS166941

Noggin Protects against Ischemic Brain Injury in Rodents

Jayshree Samanta, MBBS PhD,1,3,4,* Tord Alden, MD,2,4 Kevin Gobeske, MPH,1 Lixin Kan, MD PhD,1 and John A Kessler, MD1,*

Abstract

Background and Purpose

Bone Morphogenetic Proteins (BMPs) as well as their receptors are expressed in adult brain, and their expression levels increase following cerebral ischemia. The brain also expresses an inhibitor of BMP signaling, noggin, but the role of noggin in ischemic disease outcome has not been studied.

Methods

We used transgenic mice overexpressing noggin (NSE-noggin) to assess whether inhibition of BMP signaling affects ischemic injury responses following permanent middle cerebral artery occlusion (pMCAO).

Results

NSE-noggin mice had significantly smaller infarct volumes and lower motor deficits compared to wildtype mice. CD11b+ and IBA1+ microglia along with oligodendroglial progenitors were significantly increased in NSE-noggin mice at 14 days following pMCAO.

Conclusions

These results provide genetic evidence that overexpression of noggin reduces ischemic brain injury following pMCAO via enhanced activation of microglia and oligodendrogenesis.

Keywords: noggin, stroke, microglia, oligodendrocytes, BMP

Stroke is one of the leading causes of death and disability worldwide, and identification of new therapeutic agents is a major health imperative. Several studies of bone morphogenetic proteins (BMPs) in ischemic rodent brains have suggested that this family of factors may play an important role in ischemic disease outcome. BMPs belong to the transforming growth factor-β (TGF-β) superfamily and play important roles in cell specification, neuronal migration, survival and dendritic development. BMPs act by binding to heterodimeric serine-threonine kinase receptors on the cell surface formed by BMPRI and BMPRII which are widely expressed in the adult brain1. BMP family members are also expressed in the adult brain with differing distributions for various family members. BMP-5 and BMP-6 are expressed in hippocampus and neocortex BMP-5 in cerebellum, BMP-5 and BMP-7 in striatum, BMP-6 and BMP-7 in meninges and choroid plexus and BMP-4 in cortex, hippocampus and cerebellum1-3. Endogenous BMP-7 as well as the BMPR-II receptor are up-regulated following transient ischemia in rodents while BMP-2/4 expression decreases in ischemic astrocytes in vitro.4,5 Administration of BMP-6 or BMP-7 in the brain prior to transient ischemia and reperfusion reduces infarct area and neurological deficit but has no effect on stroke volume when administered after ischemia5-7. Pretreatment of rodents with retinoic acid has beneficial effects on stroke outcome that is prevented by inhibition of BMP signaling8. However BMP2/4 are the major family members in cortex and hippocampus, and BMP signaling in the adult brain inhibits both neurogenesis and oligodendrogliogenesis9. This suggested that inhibition of BMP signaling after cerebral ischemia might have an overall beneficial effect. To address this possibility we analyzed the effects of stroke in transgenic mice overexpressing the BMP inhibitor, noggin in the nervous system.

Noggin is an extracellular BMP antagonist which binds BMP-2/4 with high affinity and thus interferes with their binding to receptors10. In adult brain, noggin is expressed in the olfactory bulb, piriform cortex, hippocampus and cerebellum and overexpression of noggin results in an increase in oligodendrocytes in the cortex as well as neural stem cells in the proliferative regions of the brain3,11,12. Our data indicate that noggin provides protection from ischemic disease by increasing the proliferation and activation of microglia as well as by enhanced repair of white matter.

Materials and Methods

Animals

The generation of the neuron-specific enolase (NSE)–Noggin transgenic mice, has been described previously3,12,13. NSE-noggin mice were generated in FEV1 background and backcrossed to C57BL/6 mice for at least 5 generations. All the mice were housed in a facility with a 12h light/dark cycle and allowed access to food and water ad libitum. Experiments were conducted according to protocols approved by Institutional Animal Care and Use Committee and Northwestern Center for Comparative Medicine.

Focal Cerebral ischemia

Four to five month-old male mice were used for this study. Following anesthesia (Avertin, 240 mg/kg, intraperitoneally), cortical brain ischemia was induced in accordance with the method described elsewhere14. Briefly, an incision was made in the midline on the neck. The right common carotid artery was dissected and ligated with a 4-0 silk suture. After this wound was closed, an incision was made between right ear and eye. The skull was exposed and a craniotomy was done along the length of the middle cerebral artery (MCA). The MCA was coagulated with a cauterizer (World Precision Instrument, FL, USA) distal to the striatal branch under a microscope. In mice undergoing sham surgery, the MCA was exposed but not cauterized. After irrigating and closing the wound, the mice recovered from anaesthesia on a heating blanket. They were given 1ml bolus of lactated ringer’s solution mixed with buprenex 2mg/kg after surgery and then daily for 2 days.

Accelerated rotarod test

To analyze balance and coordination, all mice were trained and familiarized on the Rotarod treadmill (Med associates; St. Albans, VT) five times a day for 12 days before induction of infarct. Mice were placed on the rod rotating at 4 rpm between dividers. The accelerated speed was started and allowed to run until each mouse fell and disrupted the infrared beam located at the bottom of the Rotarod. The Rotarod automatically recorded the length of time that each mouse was able to stay on the rotating rod. The Rotarod test was carried out at an accelerating speed of between 4 and 40 rpm for 20 min with at least 30 min rest between runs for each mouse. Data from NSE-noggin mice were compared with those from age-matched wildtype mice. Statistical comparison between the two groups of mice was performed using Student’s t test.

Triphenyltetrazolium chloride (TTC) staining

Mice were euthanized 14 days after MCA occlusion and the brains were sectioned coronally using a mouse brain matrix. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO)15. The border between infarcted and non-infarcted tissue was marked by using NIH ImageJ. The area of infarction was measured using scion software and infarct volume was calculated blindly with a previously described semiautomated method that corrects for edema16.

Immunohistochemistry

Mice were anaesthetized and their brains were processed for immunohistochemistry as described previously17. Primary antibodies used were rat monoclonal antibodies to detect CD11b (1:500; ABD serotec, Raleigh, NC) and PDFGRα (1:200; BD Biosciences, San Jose, CA); rabbit polyclonal antibodies to detect IBA1 (1:150; Wako, Japan) and NG2 (1:200; Chemicon, Temecula, CA); mouse monoclonal antibodies to detect CNPase (1:200; Covance, Berkeley, CA); myelin basic protein (MBP; 1:500; Covance); GFAP (1:100; Sigma); Hu (1:150; Abcam, Cambridge, MA) and guinea pig polyclonal antibody to detect doublecortin (1:500; Abcam) immunoreactivity. Images were taken using Zeiss LSM 510 Meta Confocal Microscope and cell counts were done with NIH Image J software.

Results

Transgenic overexpression of Noggin reduces infarct volume

NSE-noggin transgenic mice overexpress noggin under the NSE promoter which is specifically active in terminally differentiated neurons in the brain. NSE expression begins as early as embryonic day 12.5 (E12.5) and continues in adult rodents18,19. We produced focal cerebral ischemia in adult NSE-noggin and wildtype mice by pMCAO. We sacrificed the mice 14 days after pMCAO or sham surgery and delineated the infarct area by TTC staining (Fig. 1 A-C). The NSE-noggin mice showed a significant reduction in the infarct volume as compared to wildtype mice (p = 0.006, n=3, Fig. 1 D).

Figure 1
Transgenic overexpression of Noggin reduces infarct volume induced by focal cerebral ischaemia in mice. Infarct volumes were calculated 14 days following permanent MCAO by TTC staining (A-C). NSE-noggin mice showed a reduction in infarct volume (D). Motor ...

Overexpression of Noggin protects motor function

To assess motor function we used the accelerated rotarod test and trained the mice before pMCAO until their performance reached a plateau. Following pMCAO we observed that NSE-noggin mice did not show any motor deficit as compared to pre-infarct levels. This was in contrast to wildtype mice which showed significant motor deficit 24 hours after pMCAO (p = 0.03, paired T-test, Fig. 1 E) but recovered to pre-pMCAO level by 5 days after pMCAO. We confirmed that the MCA was cauterized in the NSE-noggin mice by performing micro-CT (computerized tomography) with a vascular contrast agent one day following pMCAO (data not shown). This suggests that overexpression of noggin in the brain provides enhanced tolerance to ischemic injury.

Noggin increases the numbers of activated microglia in the peri-infarct area

Tolerance to ischemic brain injury can be induced by preconditioning with lipopolysaccharide (LPS) which is known to activate microglia20,21, and administration of exogenous microglia during MCAO also ameliorates ischemic damage and reduces infarct volume22,23. We therefore asked whether the beneficial effects of noggin reflected an alteration in the microglial response. At 14 days after pMCAO, we observed that NSE-noggin mice showed significantly higher numbers of CD11b+ microglia in the contralateral cortex (p=0.05, T-test, Fig. 2 A-B, E). The ischemic boundary zone (IBZ) of NSE-noggin mice also showed higher numbers of CD11b+ microglia compared to wildtype mice (Fig. 2 C-D, E) and a significant increase in numbers of IBA1+ activated microglia (p=0.02, T-test, Fig. 3 A-E). These results suggest that overexpression of noggin leads to proliferation of microglia in both hemispheres along with an enhancement of microglial activation in the ipsilateral hemisphere following ischemic injury.

Figure 2
Overexpression of noggin results in an increase in number of microglia in the uninjured brain. Microglia were labelled with CD11b in wildtype and NSE-noggin 14 days after MCAO (A-D) and positive cells were counted in the uninjured cortex as well as the ...
Figure 3
Noggin increases the numbers of activated microglia in the peri-infarct area. Immunohistochemistry for IBA1 was done to examine activated microglia (A-D) in wildtype and NSE-noggin mice 14 days after MCAO. NSE-noggin mice showed higher numbers of IBA1+ ...

Noggin increases the number of oligodendroglial progenitors in the IBZ

Transgenic overexpression of BMP2/4 increases astrogliosis and decreases oligodendrogenesis which are both reversed by noggin12. Since the cortical white matter is susceptible to ischemic injury, we examined whether overexpression of noggin resulted in alteration of oligodendroglial cells24. The IBZ of NSE-noggin mice contained cells expressing PDGFRα, the earliest marker of committed oligodendroglial progenitor cells (OPCs), whereas these cells were not observed in wildtype mice (Fig 4 A-D). There were also fewer CNPase+ and MBP+ oligodendrocytes in the peri-infarct area of NSE-noggin mice as compared to wildtype mice (Fig. 4 E-H). This suggests that overexpression of noggin produces more OPCs in response to ischemic injury. In order to assess whether the increase in OPCs along with microglial activation is associated with enhanced neurogenesis, we counted the number of Doublecortin+ (DCX) neuroblasts and Hu+ neurons. There were no significant differences in the numbers of DCX+ (Fig. 5 A-B) or Hu+ (Fig. 5 C-D) cells in the IBZ of NSE-noggin mice compared to wildtype mice. Finally we examined if noggin affects the formation of glial scar following pMCAO. NSE-noggin mice did not show any difference in number of NG2+ cells which produce chondroitin sulfate proteoglycans as compared to wildtype IBZ (Fig. 5 A-B). We also compared the GFAP+ area surrounding the infarct and did not observe any significant difference between the transgenic and wildtype mice (Fig. 6 C-F). These results indicate that overexpression of noggin specifically enhances the repair of white matter injury following ischemia.

Figure 4
Noggin increases the number of oligodendroglial progenitors in the IBZ. Oligodendroglial precursor cells labelled with PDGFRα were only detected in the IBZ of NSE-noggin mice (A,B). There were fewer numbers of CNPase+ and MBP+ cells in the NSE-noggin ...
Figure 5
Noggin does not affect the number of neurons in the peri-infarct area. Immunohistochemistry was done to label immature neurons with Doublecortin (A,B) and all neurons with Hu (C,D). Labelled cells were counted in the IBZ of NSE-noggin and wildtype mice ...
Figure 6
Transgenic overexpression of noggin does not affect the formation of glial scar. NG2+ cells were counted in the IBZ of NSE-noggin and wildtype and no significant difference was detected (A,B). No significant difference was found in the area of GFAP+ astrocytes ...

Discussion

We have shown that overexpression of noggin in the brain provides protection and/or enhanced recovery from ischemic injury following pMCAO via enhanced activation of microglia and oligodendrogenesis. It is possible that noggin also activates other protective mechanisms. In human patients, activation of microglia increases after 72 hours following stroke and inflammation persists in both hemispheres up to 30 days25. Further, Iba1 expression in activated microglia increases during the first 3 days post-infarct, and remains elevated in chronic stages26. After pMCAO in rodents, activated resident microglia, along with hematogenous macrophages infiltrating the parenchyma, continue to increase in numbers up to day 14 when the debris removal reaches its peak27. The role of microglia in stroke is controversial and different studies have given contradicting results. Most studies suggesting that microglia have neurotoxic effects have been done in transient MCAO with a reperfusion stroke model. Thus, null mutation of CD11b knock-out reduces stroke volume following transient MCAO after 24 hrs indicating that microglia are neurotoxic at that stage28,29. However, at 72 hours following transient MCAO, selective ablation of CD11b expressing microglia results in an increase in infarct volume along with neuronal apoptosis suggesting that microglia are neuroprotective30. These divergent findings suggest that the role of microglia changes at distinct temporal phases following MCAO.

Our studies of the microglial response 14 days following ischemia in a permanent model suggest that microglia have beneficial effects on neurological function and infarct volume (Fig. (Fig.11--2).2). Our results also support other studies demonstrating that administration of exogenous microglia protect against neurodegeneration22,23. The mechanism of neuroprotection by microglia has been attributed in some studies to the production of insulin like growth factor-1 (IGF1) by activated microglia30,31. IGF1 also promotes the differentiation of adult neural stem cells into oligodendrocytes by inhibiting BMP-2/4 signaling and addition of noggin has a synergistic effect with IGF1 on production of oligodendrocytes32. In our study we found the presence of OPCs in the peri-infarct area of NSE-noggin mice (Fig. 4), consistent with the idea that noggin and IGF1 produced by activated microglia act synergistically to repair the white matter injury following stroke.

While administration of BMP-6 and BMP-7 have been shown to improve neurological function and reduce stroke volume in transient MCAO models, our results show that more global inhibition of BMP signaling with noggin is beneficial in a pMCAO model. In addition to differences in the stroke model and timing of analysis, this could be explained by the fact that noggin binds BMP-2/4 more avidly than BMP-6 and BMP-7; thus our results are probably due to the predominant inhibition of BMP-2/4 effects. BMP-2 and BMP-7 can exert opposite effects during development, and our findings may reflect the same divergence in function of different BMP family members in adults33.

In our study, overexpression of noggin protected the mice from neurological deficit a day after pMCAO suggesting that noggin is neuroprotective. Noggin was present at the time of the stroke, so further studies must be done to assess the therapeutic potential of administering exogenous noggin following ischemia. The incidence of stroke depends upon genetic and environmental risk factors in different human populations; for example, certain Mediterranean populations have a significantly lower incidence34. Further genetic studies may shed light on whether noggin polymorphisms confer protection from stroke in human populations.

Acknowledgements

This work was supported by NIH grants NS 20013 and NS 20778.

Footnotes

Conflict of Interest None

References

1. Charytoniuk DA, Traiffort E, Pinard E, Issertial O, Seylaz J, Ruat M. Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia. Neuroscience. 2000;100:33–43. [PubMed]
2. Harvey BK, Hoffer BJ, Wang Y. Stroke and TGF-beta proteins: glial cell line-derived neurotrophic factor and bone morphogenetic protein. Pharmacol Ther. 2005;105:113–25. [PubMed]
3. Bonaguidi MA, Peng CY, McGuire T, Falciglia G, Gobeske KT, Czeisler C, Kessler JA. Noggin expands neural stem cells in the adult hippocampus. J Neurosci. 2008;28:9194–204. [PMC free article] [PubMed]
4. Xin H, Li Y, Chen X, Chopp M. Bone marrow stromal cells induce BMP2/4 production in oxygen-glucose-deprived astrocytes, which promotes an astrocytic phenotype in adult subventricular progenitor cells. J Neurosci Res. 2006;83:1485–93. [PMC free article] [PubMed]
5. Chang C-F, Lin S-Z, Chiang Y-H, Morales M, Chou J, Lein P, Chen H-L, Hoffer BJ, Wang Y. Intravenous Administration of Bone Morphogenetic Protein-7 After Ischemia Improves Motor Function in Stroke Rats. 2003:558–564. [PubMed]
6. Chang CF, Morales M, Chou J, Chen HL, Hoffer B, Wang Y. Bone morphogenetic proteins are involved in fetal kidney tissue transplantation-induced neuroprotection in stroke rats. Neuropharmacology. 2002;43:418–26. [PubMed]
7. Wang Y, Chang CF, Morales M, Chou J, Chen HL, Chiang YH, Lin SZ, Cadet JL, Deng X, Wang JY, Chen SY, Kaplan PL, Hoffer BJ. Bone morphogenetic protein-6 reduces ischemia-induced brain damage in rats. Stroke. 2001;32:2170–8. [PubMed]
8. Shen H, Luo Y, Kuo CC, Deng X, Chang CF, Harvey BK, Hoffer BJ, Wang Y. 9-Cis-retinoic acid reduces ischemic brain injury in rodents via bone morphogenetic protein. J Neurosci Res. 2009;87:545–55. [PMC free article] [PubMed]
9. Colak D, Mori T, Brill MS, Pfeifer A, Falk S, Deng C, Monteiro R, Mummery C, Sommer L, Gotz M. Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells. J Neurosci. 2008;28:434–46. [PubMed]
10. Zimmerman LB, De Jesus-Escobar JM, Harland RM. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell. 1996;86:599–606. [PubMed]
11. Valenzuela DM, Economides AN, Rojas E, Lamb TM, Nunez L, Jones P, Lp NY, Espinosa R, 3rd, Brannan CI, Gilbert DJ, et al. Identification of mammalian noggin and its expression in the adult nervous system. J Neurosci. 1995;15:6077–84. [PubMed]
12. Gomes WA, Mehler MF, Kessler JA. Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment. Dev Biol. 2003;255:164–77. [PubMed]
13. Kan L, Hu M, Gomes WA, Kessler JA. Transgenic mice overexpressing BMP4 develop a fibrodysplasia ossificans progressiva (FOP)-like phenotype. Am J Pathol. 2004;165:1107–15. [PMC free article] [PubMed]
14. Coyle P. Middle cerebral artery occlusion in the young rat. Stroke. 1982;13:855–9. [PubMed]
15. Isayama K, Pitts LH, Nishimura MC. Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke. 1991;22:1394–8. [PubMed]
16. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab. 1990;10:290–3. [PubMed]
17. Samanta J, Burke GM, McGuire T, Pisarek AJ, Mukhopadhyay A, Mishina Y, Kessler JA. BMPR1a signaling determines numbers of oligodendrocytes and calbindin-expressing interneurons in the cortex. J Neurosci. 2007;27:7397–407. [PubMed]
18. Forss-Petter S, Danielson PE, Catsicas S, Battenberg E, Price J, Nerenberg M, Sutcliffe JG. Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control. Neuron. 1990;5:187–97. [PubMed]
19. Forss-Petter S, Danielson P, Sutcliffe JG. Neuron-specific enolase: complete structure of rat mRNA, multiple transcriptional start sites, and evidence suggesting post-transcriptional control. J Neurosci Res. 1986;16:141–56. [PubMed]
20. Rosenzweig HL, Lessov NS, Henshall DC, Minami M, Simon RP, Stenzel-Poore MP. Endotoxin preconditioning prevents cellular inflammatory response during ischemic neuroprotection in mice. Stroke. 2004;35:2576–81. [PubMed]
21. Ahmed SH, He YY, Nassief A, Xu J, Xu XM, Hsu CY, Faraci FM. Effects of lipopolysaccharide priming on acute ischemic brain injury. Stroke. 2000;31:193–9. [PubMed]
22. Kitamura Y, Takata K, Inden M, Tsuchiya D, Yanagisawa D, Nakata J, Taniguchi T. Intracerebroventricular injection of microglia protects against focal brain ischemia. J Pharmacol Sci. 2004;94:203–6. [PubMed]
23. Hayashi Y, Tomimatsu Y, Suzuki H, Yamada J, Wu Z, Yao H, Kagamiishi Y, Tateishi N, Sawada M, Nakanishi H. The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats. Neuroscience. 2006;142:87–96. [PubMed]
24. Pantoni L, Garcia JH, Gutierrez JA. Cerebral white matter is highly vulnerable to ischemia. Stroke. 1996;27:1641–6. discussion 1647. [PubMed]
25. Price CJ, Wang D, Menon DK, Guadagno JV, Cleij M, Fryer T, Aigbirhio F, Baron JC, Warburton EA. Intrinsic activated microglia map to the peri-infarct zone in the subacute phase of ischemic stroke. Stroke. 2006;37:1749–53. [PubMed]
26. Postler E, Rimner A, Beschorner R, Schluesener HJ, Meyermann R. Allograft-inflammatory-factor-1 is upregulated in microglial cells in human cerebral infarctions. J Neuroimmunol. 2000;104:85–91. [PubMed]
27. Stoll G, Jander S, Schroeter M. Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol. 1998;56:149–71. [PubMed]
28. Soriano SG, Coxon A, Wang YF, Frosch MP, Lipton SA, Hickey PR, Mayadas TN. Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury. Stroke. 1999;30:134–9. [PubMed]
29. Arumugam TV, Salter JW, Chidlow JH, Ballantyne CM, Kevil CG, Granger DN. Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion. Am J Physiol Heart Circ Physiol. 2004;287:H2555–60. [PubMed]
30. Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. Selective Ablation of Proliferating Microglial Cells Exacerbates Ischemic Injury in the Brain. J Neurosci. 2007:2596–2605. [PubMed]
31. O’Donnell SL, Frederick TJ, Krady JK, Vannucci SJ, Wood TL. IGF-I and microglia/macrophage proliferation in the ischemic mouse brain. Glia. 2002;39:85–97. [PubMed]
32. Hsieh J, Aimone JB, Kaspar BK, Kuwabara T, Nakashima K, Gage FH. IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes. J Cell Biol. 2004;164:111–22. [PMC free article] [PubMed]
33. Michon F, Forest L, Collomb E, Demongeot J, Dhouailly D. BMP2 and BMP7 play antagonistic roles in feather induction. Development. 2008;135:2797–805. [PMC free article] [PubMed]
34. Manobianca G, Zoccolella S, Petruzzellis A, Miccoli A, Logroscino G. Low incidence of stroke in southern Italy: a population-based study. Stroke. 2008;39:2923–8. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...