Axonal Outgrowth and Dendritic Plasticity in the Cortical Peri-Infarct Area After Experimental Stroke

Introduction

Brain injury including stroke induces limited axonal regrowth. Emerging data indicate that axonal remodeling is a critical aspect of brain repair and contributes to spontaneous improvements of neurological deficits after stroke.^1,2

Neurofilament (NF), a neuron-specific intermediate filament, is the most abundant architectural cytoskeletal element in axons and dendrites.³ Neurofilaments are composed of 3 different subunits, light (68kDa), medium (150 kDa), and heavy (NFH, 200 kDa).³ The activity of NF depends on its state of phosphorylation.⁴ Phosphorylated NFH (pNFH) participates in axonal growth and regulates synaptic function.^5,6 Several articles have suggested that aberrant perikaryal accumulation hyperphosphorylated NFH in neurodegenerative disorders.^7,8 Nonphosphorylated NFH (npNFH) is also described as being more abundant in regenerating axons of injured nerves, whereas npNFH indicates damaged and demyelinated axons in multiple sclerosis.^9,10 Until recently, only a few studies have examined changes in pNFH after stroke. Stroke induces pNFH in perikarya of injured neurons in human brain.¹¹ In the rodent, cortical infarct results in reduction of pNFH within the peri-infarct region.¹² Mechanisms that regulate axonal regrowth and phosphorylation of NFH after stroke have not been fully investigated.

In the present study, we analyzed the profiles of axonal outgrowth and myelination in rat ischemic brains. We also investigated axonal outgrowth and the signaling pathways that mediate axonal outgrowth in cultured cortical neurons.

Methods

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

Focal Cerebral Ischemia

Adult male Wistar rats (≈350 g, Charles River) were subjected to permanent right middle cerebral artery occlusion (MCAO) by advancing a 4 to 0 surgical nylon suture with an expanded tip.^2,13 Rats were euthanized at 7, 28, or 56 days after MCAO. Peri-infarct area in cerebral cortex of the rat was defined on Hematoxylin and Eosin-stained coronal sections (8 μm) as the area encompassed by a 300 μm distance from the infarction.¹⁴

Primary Cortical Neurons

Cortical neurons were harvested from embryonic day-17 Wistar rats (Charles River), according to a published protocol. To separate axons from neuronal soma, a microfluidic chamber (Standard Neuron Device, Xona Microfluidics) was employed.¹⁵ Oxygen-glucose deprivation (OGD) was performed for 3 hours. See Supplemental Methods.

Coculture of Neurons With Oligodendrocyte Cells

Standard coculture system was employed according to published protocols. Briefly, mouse premature oligodendrocyte cells (N20.1, generously provided by Dr. Anthony Campagnoni, University of California at Los Angeles) were incubated in DMEM/F12 at 39°C for 9 days.¹³ See Supplemental Methods.

Experimental Protocol

To examine the effect of Akt or (glycogen synthase kinase-3β) GSK-3β on axonal outgrowth and pNFH expression after ischemia, neurons were treated with pharmacological phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, 10, 20, and 40 μmol/L; Calbiochem) and Wortmannin (2 μmol/L; Calbiochem), or GSK-3 inhibitors, inhibitor I (TDZD-8, Thiadiazolidinone analog, 5 μmol/L; Calbiochem), and inhibitor VII (α-4-dibromoacetophenone, 1 μmol/L; Calbiochem) for 96 hours after OGD.^16–18

Immunohistochemistry and Immunocytochemistry

Single and double immunofluorescent staining was performed on brain sections and cultured cells, as previously described.¹⁶ See Supplemental Methods.

Golgi-Cox Staining

Golgi-Cox staining was performed on brain tissue according to a protocol kindly provided by Dr Crystal L. MacLellan.¹⁹

Image Acquisition and Quantification

For histological study, 3 coronal sections at 200 μm intervals/rat per staining were used. Three images of peri-infarct area in cerebral cortex per section were acquired using a 2-photon microscope (Zeiss LSM 510 NLO) under a 40× objective. Images were analyzed using the National Institutes of Health Image analysis program version 1.43, as described previously.²⁰ See Supplemental Methods.

Western Blot

Western blots were performed according to published methods.¹⁶ See Supplemental Methods.

Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on an ABI 7000 PCR instrument (Applied Biosystems) using SYBR Green real-time PCR method.¹⁶ See Supplemental Methods.

Time-Lapse Microscopy

Axons cultured in microfluidic chamber were incubated in a stage top chamber with 5% CO₂ at 37°C, which was placed on the stage of a TE2000-U inverted microscope equipped with a motorized z-stage (Nikon). See Supplemental Methods.

Statistical Analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences (version 15; SPSS Inc). Student t test and 1-way ANOVA with post hoc Bonferroni were used when comparing 2 groups and more than 2 groups, respectively. Values presented in this study are expressed as mean±standard error. A probability value <0.05 was considered significant.

Results

NFH Immunoreactivity in Experimental Stroke

Neurons express pNFH and npNFH in their axons and dendrites.⁵ We found that pNFH immunoreactive neuronal fibers were closely associated with myelin basic protein (MBP, a marker of myelinating oligodendrocytes) immunoreactive processes in the cortical layer of sham-operated rats (Supplemental Figure S1A); and approximately, 70% of pNFH⁺ fibers were associated with MBP in layer VI (Supplemental Figure S1A). This suggests that the majority of pNFH⁺ fibers are myelinated axons. The npNFH⁺ fibers were also adjacent to MBP⁺ processes in layer VI, although npNFH were expressed in neuronal soma and apical dendrites in layer II to V (Supplemental Figure S1B). The pNFH⁺ axons and npNFH⁺ axons, soma, and dendrites, substantially decreased in axonal and somal compartments in the peri-infarct area after stroke (Supplemental Figure S2A). In layer VI of rat cerebral cortex, MCAO resulted in a well-demarcated infarction in the territory supplied by the MCA during 7 to 56 days poststroke (Figure 1A). We measured pNFH immunoreactive fibers in the peri-infarct area (Figure 1A) and found a significant reduction of pNFH⁺ fibers of up to 63% at 7 days of MCAO in the peri-infarct area compared with homologous areas of sham-operated rats (Figure 1B, C). However, pNFH⁺ fibers in the peri-infarct area increased to 81% and 97% of intact levels at 28 and 56 days after MCAO, respectively (Figure 1B, C). Moreover, the number of NeuN⁺ cells in the area did not significantly change during this period (Figure 1B). Interestingly, in the contralateral cortex, pNFH⁺ fibers substantially decreased to 68% at 7 days after MCAO compared with in sham-operated rats, and increased to 79% and 95% of intact levels at 28 and 56 days after MCAO, respectively (Supplemental Figure S2B). In addition, at 7 days of MCAO, pNFH⁺ fibers associated with MBP⁺ processes were reduced by 60% in the peri-infarct area compared with homologous areas of sham-operated rats (Figure 1B, D); whereas at 56 days after MCAO, pNFH⁺ fibers surrounded by MBP immunoreactivity increased to 98% of intact levels, which is equivalent to the levels of pNFH and MBP in sham-operated rats (Figure 1B, D). A comparable profile of npNFH immunoreactive fibers was also detected (Supplemental Figure S2C, S3A-C). These data suggest that myelinated axons are increased in the peri-infarct area during stroke recovery.

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Axonal outgrowth is closely related to growth of dendrites and dendritic spines.²¹ At 7 days following MCAO, Golgi-Cox staining indicated that neurons showed a marked deterioration of basilar dendrites with connection to adjacent neurons, dramatic reduction of the number and diameter of dendrites, and reduction in spine number of apical dendrites compared with neurons in sham-operated rats (Figure 2A, B). Moreover, swollen dendritic spines increased (Figure 2B, Supplemental Figure S4A). However, number of spines in apical dendrites significantly increased at 56 days compared with 7 days after MCAO (Figure 2C, Supplemental Figure S4B). These dendrites increased in number and length and they connected to dendrites derived from adjacent neurons (Figure 2C, Supplemental Figure S4C, D), whereas only scattered swollen spines were present (Supplemental Figure S4A).

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pNFH in Cultured Cortical Neurons

Aforementioned in vivo data suggest that regeneration of axons occurs in the peri-infarct area. To examine directly axonal regeneration and sprouting, we employed a microfluidic chamber, which separates axons from neuronal cell bodies and permits direct axonal outgrowth monitoring in cortical neurons.¹⁵ Cortical neurons cultured in a microfluidic chamber exhibited axonal morphology (Figure 3A). OGD for 3 hours did not significantly increase caspase-3 levels in cortical neurons (Supplemental Figure S5A), but induced damaged axons with beaded and vanishing appearance at 24 hours (Figure 3A, Supplemental Figure S5B). However, 96 hours after OGD, a large number of axons were regenerated (Figure 3A, B). RT-PCR and Western blot analysis showed that mRNA levels of NEFH and protein levels of pNFH substantially increased at 96 hours compared with 24 hours after OGD, as well as in control non-OGD neurons (Figure 3C, Supplemental Figure S5C). To examine whether these axons can be myelinated, we cocultured axons with differentiated N20.1 cells in the axonal compartment of the microfluidic chamber. Double immunostaining revealed that many pNFH⁺ axons were surrounded by 2′, 3′-cyclic nucleotide 3′-phosphodiesterase⁺ oligodendrocyte processes at 96 hours after OGD (Figure 3D). Collectively, these data suggest that OGD induces axonal regeneration and sprouting, and newly generated axons can be myelinated by oligodendrocytes in vitro.

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Phosphorylation of GSK-3β Enhances pNFH and Axonal Growth

Several signaling pathways, including PI3K/Akt signaling, mediate growth of axonal and dendritic branches.²² Western blot analysis showed a significant increase in pAkt in neurons at 96 hours after OGD compared with neurons without OGD, which was coincident with elevation of pGSK-3β Ser9 (Figure 4A). These data suggest that the activation of PI3K/Akt phosphorylates GSK-3β at Ser9. To examine whether stroke induces pGSK-3β, we performed immunostaining on brain coronal sections. Double immunofluorescent staining revealed that pNFH⁺ processes in peri-infarct areas were pGSK-3β⁺ (Figure 4B), suggesting activation of inhibitory GSK-3β in vivo. To examine further the connection between PI3K/Akt activity and phosphorylation of GSK-3β, we treated neurons subjected to OGD with PI3K inhibitors, LY294002 and Wortmannin^16,17; both inhibitors significantly decreased pAkt and reduced pGSK-3β Ser9 (Figure 4C, Supplemental Figure S6B, C). To test whether increases in the levels of pGSK-3β that inactivate GSK-3β are functionally relevant to elevated pNFH, we blocked GSK-3β activity using 2 structurally unrelated non-ATP competitive GSK-3 inhibitors, inhibitor I and VII.¹⁸ Treatment of OGD-challenged neurons with both GSK-3 inhibitors further elevated pGSK-3β Ser9 and pNFH, whereas PI3K inhibitors substantially decreased pNFH protein levels (Figure 4C, Supplemental Figure S6A, C). In parallel, time-lapse microscopy revealed that inhibition of GSK-3β activation by GSK-3 inhibitor I increased axonal elongation (Figure 5A, C) and pNFH⁺ arborization (Figure 5B, D). In contrast, a PI3K inhibitor, LY294002, significantly suppressed axonal outgrowth (Figure 5A, C) and pNFH⁺ arborization (Figure 5B, D).

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LY294002 (40 μmol/L) did not significantly induce cell death measured by TUNEL positive cells (Supplemental Table S1), and did not significantly increase the number of damaged axons compared with neurons without treatment after OGD (Supplemental Figure S6D). Consistent with concentration of 40 μmol/L, we found that LY294002 at 10 and 20 μmol/L significantly (P<0.01) blocked axonal elongation by 30% and 43%, respectively (n=80 axons/group), compared with the control group (n=70 axons/group).

Phosphatase tensin homolog deleted on chromosome 10 (PTEN) inhibits Akt activity and mediates axonal outgrowth.²³ Cyclin-dependent kinase regulates axonal transport and phosphorylates NFH.²⁴ Axons at 96 hours after OGD exhibited a substantial reduction of phosphorylated PTEN (Figure 4D), but levels of total and phosphorylated cyclin-dependent kinase did not significantly change compared with levels in the control group (data not shown); this suggests that downregulation of PTEN activates Akt, whereas cyclin-dependent kinase may not play an important role in OGD-elevated pNFH. Collectively, our data suggest that inactivation of GSK-3β by its phosphorylation at Ser9 enhances axonal outgrowth via the PI3/Akt pathway in OGD-challenged neurons.

Discussion

The present study demonstrates that in rodent ischemic brain, stroke substantially increased NFH⁺ axons in peri-infarct cortex and in homologous areas of the contralateral cortex during the recovery period, and increased NFH⁺ axons were closely associated with myelinating oligodendrocytes. Moreover, in vitro data show that inhibition of GSK-3β elevated pNFH levels and enhanced axonal outgrowth of the cortical neurons challenged by OGD via the PI3/Akt pathway. These data suggest that the PI3K/Akt/GSK-3β signaling pathway could be a potential therapeutic target for promoting axonal regrowth after stroke.

Stroke acutely causes a loss of pNFH⁺ axons in mammals.¹² In human brains after stroke, pNFH accumulates in perikarya and dendrites.¹¹ However, little information is available on the distribution of pNFH⁺ axons in ischemic brain during stroke recovery. Our experimental data revealed that acute stroke induced loss in NFH⁺ axons. More importantly, we found gradual, but substantial, increases in NFH⁺ axons in the peri-infarct cortex, and many of these axons were surrounded by MBP⁺ processes during stroke recovery; this suggests that stroke induces axonal regrowth and newly generated axons are myelinated. Likewise, in the contralateral cortex, we detected a substantial reduction and subsequent increase of pNFH⁺ axons in acute stroke and recovery from stroke, respectively. This finding is consistent with recently published studies showing an increase in axonal outgrowth in the contralateral cortex during stroke recovery in the rodent, which was facilitated by treatment with inosine or bone marrow stromal cells.^2,25

A major challenge for studying axonal outgrowth of cultured cortical neurons is the need to isolate axons from other cellular debris, which impedes investigation of mechanisms underlying axonal outgrowth. We employed a recently developed microfluidic platform that isolates axons from dendrites of cortical neurons.¹⁵ We demonstrated that cortical neurons injured by OGD have elevated pNFH levels and regenerate axons, which was associated with increases in phosphorylation of GSK-3β. Moreover, inhibition of GSK-3β with pharmacological inhibitors increased phosphorylation of GSK-3β and pNFH, and it promoted axonal regeneration and arborization. Phosphorylation of GSK-3β inactivates GSK-3β.²⁶ GSK-3β acts as a negative regulator of axon formation.²⁷ Phosphorylated GSK-3β has been implicated in the phosphorylation of NFH,²⁸ given that GSK-3β has affinity to the NFH side arm.²⁹ Thus, our data for the first time indicate that in cortical neurons, pGSK-3β mediates pNFH expression and axonal regeneration after OGD. However, in addition to neurons, GSK-3β plays important roles in brain parenchymal cells, including in neural stem cells. Administration of GSK-3β inhibitors will not selectively block neuronal GSK-3β activity in vivo. Investigation of the role of GSK-3β in axonal outgrowth after stroke by use of a conditional GSK-3β mouse (Gsk-3β^flox/flox) to create mice with neuron-specific GSK-3β deficiency is warranted.

PI3K activates Akt.²² On activation, Akt phosphorylates diverse substrates, including GSK-3β.²² PTEN inhibits PI3K/AKT signaling.²⁷ Activation of Akt and subsequent phosphorylatory inhibition of GSK-3β increases axonal outgrowth of dorsal root ganglial neurons.³⁰ The present study demonstrates that elevation of pNFH in cortical axons was associated with downregulation of PTEN and activation of Akt, whereas inhibition of PI3K suppressed Akt activation and downregulated phosphorylation of GSK-3β. Therefore, our data indicate that reduction of PTEN activates PI3K/Akt signaling, which in turn inhibits GSK-3β by its phosphorylation, leading to axonal outgrowth of cortical neurons after OGD. A caveat is that these in vitro findings cannot be directly applied to in vivo observation,³¹ However, our in vivo immunohistochemistry data suggest the activation of inhibitory GSK-3β in pNFH fibers in the peri-infarct area. Others have demonstrated that deletion of PTEN enhances regeneration of adult corticospinal tract in mouse model of spinal cord injury.²³ Therefore, inhibition of GSK-3β activity in axons has a potential therapeutic effect to enhance axonal generation in the adult injured brain after stroke.

Sources of Funding

This work was supported by the National Institutes of Health (PO1 NS23393, RO1 AG037506, and RO1 NS75156).

Disclosures

None.

Footnotes