Brain Res. 2015 Apr 24;1605:76-82.
Changes in the BDNF-immunopositive cell population of neocortical layers I and II/III after focal cerebral ischemia in rats
Yongwon Choia,d, Sung Goo Kangb, Kyung-Yoon Kamc,d,*
aDepartment of Rehabilitation Science, Inje University, Gimhae 50834, Republic of Korea
bDepartment of Biological Sciences, Institute of Basic Science, Inje University, Gimhae 50834, Republic of Korea
cDepartment of Occupational Therapy, Inje University, Gimhae 50834, Republic of Korea
dU-Healthcare & Anti-aging Research Center, Inje University, Gimhae 50834, Republic of Korea
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is widely distributed in the central nervous system, including the cerebral cortex. BDNF plays an important role in normal neural development, survival of existing neurons, and activity- dependent neuroplasticity. BDNF can also be neuroprotective and evoke neurogenesis in certain pathological conditions, such as cerebral ischemia. Neocortical layer I is an important region that can integrate feedforward and feedback information from other cortical areas and subcortical regions. In addition, it has recently been proposed as a possible source of neuronal progenitor cells after ischemia. Therefore, we investigated changes in the BDNF- immunorecative cell population of neocortical layers I and II/III after middle cerebral artery occlusion (MCAO)-induced cerebral ischemia in rats. In unaffected condition, the number of BDNF+ cells in layer I was significantly less than in layer II/III in the cingulate cortex and in the motor and sensory areas. The increase in the number of BDNF+ cells in layer I 8 days after MCAO was more remarkable than layer II/III, in all regions except the area of cingulate cortex farthest from the infarct core. Only BDNF+–Ox-42+ cells showed a tendency to increase consistently toward the infarct core in both layers I and II/III, implying a major source of BDNF for response to ischemic injury. The present study suggests that some beneficial effects during recovery from ischemic injury, such as increased supportive microglia/macrophages, occur owing to a sensitive response of BDNF in layer I.
Key words: BDNF, cerebral ischemia, neocortical layer I
What does neocortical layer I do for recovery after cerebral ischemic injury?
After MCAO, ischemic lesions are commonly seen in the striatum and cortex (Figure 1). These lesions form an infarct core with severely compromised blood flow, which is surrounded by a peri-infarct area. Typically, the neocortex of the rat brain is divided into six layers. Layer I contains dendritic extensions of pyramidal neurons located in deeper layers, horizontally oriented axons, astrocytes, and only a few neurons4. However, a recent study showed that there are neural progenitor cells in layer I of the adult rat brain, which can differentiate into GABAergic interneurons in ischemia. This implies that neocortical layer I plays a key role in the recovery process this pathological state5.
We focused on neocortical layers I and II/III of regions Cg2 and Cg1 (considered intact), M2 and M1 (probably peri-infarct), and S1 (infarct) of the ischemic hemisphere. BDNF is widely distributed in the cerebral cortex and hippocampus of mammals1 and is beneficial in facilitating post-stroke recovery2, 3. Therefore, we compared changes in the number of BDNF+ cells after ischemic injury between layers I and II/III. In MCAO group, there was a significant increase in the number of BDNF+ cells in both layer I and layer II/III of all regions, except in region Cg2 (Figure 2B). As cellular components are much rarer in layer I than in layer II/III, direct comparison of cell number between these two layers did not yield a meaningful interpretation. However, the relative increase in the number of BDNF+ cells after MCAO was much higher in layer I than in layer II/III; this increase was expressed as a ratio of BDNF+ cells in MCAO versus Sham conditions. Interestingly, the ratio was greater in layer I than in layer II/III for all regions except for Cg2 that was farthest from the infarct (Figure 2C). This result suggests that layer I is more sensitive to ischemic injury, with respect to BDNF-immunoreactivity, than layer II/III. Even Cg1 that was considered an intact region showed a similar pattern.
This property of layer I should be considered carefully while investigating the mechanisms of recovery after cerebral ischemic injury. Due to the scarcity of cellular components such as neurons and glial cells, the functional significance of layer I has not been studied extensively. However, since dendritic and axonal connections in layer I receive inputs from and send outputs to cortical and subcortical regions6, respectively, this layer is an important junction where feedforward and feedback information from diverse cortical and subcortical regions could be integrated7, 8. These interconnections of layer I with other layers and subcortical structures are essential for somatosensation, cognition, and emotion6, 9, 10. In addition, increasing evidence suggests that layer I is a potential site for adult neurogenesis, especially after ischemic injury5, 11-13. Since BDNF stimulates neurogenesis, the usually quiescent local progenitor cells in neocortical layer I may be activated after ischemia, giving rise to new interneurons through a BDNF-sensitive mechanism.
This study also showed that most of the BDNF+ cells in layer I were microglia/macrophages (determined by Ox-42 immunoreactivity); although, it is difficult to differentiate between these two cell types. Since the brain samples were obtained at day 8 after MCAO and cells of these types were abundant in injured area, it was not surprised that most BDNF+ cells were these cell types. Microglial proliferation peaks at 48–72 h after focal cerebral ischemia and may last for several weeks after initial injury14. In addition, blood-derived macrophages are recruited into the ischemic brain tissue most abundantly at days 3–7 after stroke15. Therefore, this finding could also imply that increased proliferation of microglia and macrophages could be the most efficient way of supplying the neurotropic factor BDNF that is necessary for recovery after an ischemic injury.
This study conclusively suggests that neocortical layer I plays a significant role in recovery from ischemic cerebral injury by supporting neurogenesis through a BDNF-sensitive pathway. This could provide new insight into stroke therapeutics.
Figure 1. Five regions investigated after focal cerebral ischemia induced by MCAO and distributions of neurons and BDNF+ cells in layers I and II/III. (A) The schematic figure shows cingulate cortices (Cg2 and Cg1), motor cortices (M2 and M1) and sensory cortex (S1) and displays peri-infarct area (light gray) and infarct core (dark gray). (B) Immunohistochemistry for neurons (green) by using antibody against NeuN and DAPI staining (blue) for cellular nuclei in the M2 region. (C) Immunohistochemistry for BDNF (red) and DAPI staining (blue) in the same region. Cell counting was performed in a fixed-sized (600 μm x 100 μm) area (white dotted rectangle) in layers I and II/III. Small black bar marks the border between layer I and layer II/III. Red scale bar is 100 μm. Abbreviations: cingulate cortex 2 (Cg2); cingulate cortex 1 (Cg1); secondary motor cortex (M2); primary motor cortex (M1); primary somatosensory cortex (S1).
Fig. 2 More sensitive response of layer I to ischemic injury than layer II/III, with respect to BDNF-immunoreactivity. (A) Immunohistochemistry reveals BDNF+ cells (red) and DAPI staining shows cellular nuclei (blue). The upper and lower rows show BDNF+ cells of neocortical layers in Cg1, M1 and S1 regions in sham and MCAO groups respectively. White bars represent the border between layer I and layer II/III. (B) Bar graphs represent the number of BDNF+ cells as per cent of total cells in a fixed sized area (600 μm x 100 μm) in layers I and II/III of five cortical regions. (C) Bar graphs represent fold changes in the number of BDNF+ cells in layer I and layer II/III of each region after ischemic injury as MCAO/sham ratio based on the data shown in panel B. Data are expressed as mean ± S.E.M. Red scale bar is 100 μm. *p < 0.05 vs. sham group, #p < 0.05 between corresponding layers of each region.
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