Dysfunction in Motor Coordination in Neonatal White Matter Injury Model without Apparent Neuron Loss

DWMI Rat Model

Animal care was carried out according to the guidelines of the Institute for Experimental Animal Science at Nagoya City University Medical School (Nagoya, Japan). All experimental procedures were approved by the committee of animal experimentation of Nagoya City University Medical School, and every effort was made to minimize the pain and discomfort of the animals used in this study. Male neonatal Wistar rats (P3) were obtained from Japan SLC, Inc. Pups were housed with their mothers, which had access to standard chow and water ad libitum, under a constant 12-h dark/light cycle. A total of 92 pups were used for this experiment.

DWMI model rats were established according to a previously described method ²⁸ . Briefly, P3 pups (n = 65) were subjected to right common carotid artery occlusion (RCCAO) under isoflurane (Pfizer, New York, NY, USA) anesthesia [5% (v/v) induction, 1.5% (v/v) maintenance]. After a 2-h recovery period with their dam, pups were exposed to 6% (v/v) O₂ hypoxia for 60 min in a container submerged in a 38°C water bath. The pups were returned to their dam and maintained under normal conditions with a light/dark cycle of 12 h. Sham-operated rats (n = 27) were used as the control. The rats were allowed to wean at P25.

Behavioral Testing

Motor coordination was tested using the accelerating rotarod system^31,32. For habituation, 8-month-old rats (DWMI: n = 16, sham operated: n = 12) were placed on a rotating rod (10 rpm) for 5 min for 3 successive days. For the test, rats were placed on an accelerating rotarod from 4 to 40 rpm in 60 s, three times. The maximal time that the rat was able to stay on the top of the rotating rod was used for data analysis.

Immunostaining

Rats were deeply anesthetized with pentobarbital (>50 mg/kg; Kyorituseiyaku, Tokyo, Japan) and perfused with 4% (w/v) paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M of phosphate-buffered saline (PBS). Brains were postfixed overnight followed by cryoprotection in 20% (w/v) sucrose in 0.1% (v/v) PB. Serial coronal sections (20 μm) were cut and processed for histology. For immunohistochemistry, endogenous peroxidase was inactivated with 0.6% (v/v) H₂O₂ (Wako, Tokyo, Japan) dissolved in PBS + 0.1% (v/v) Triton (Nacalai Tesque, Inc., Kyoto, Japan) for 30 min. After washing with PBS, sections were blocked with 3% (v/v) serum and incubated overnight at 4°C with primary antibodies. Sections were then reacted with biotinylated secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA) and visualized using the Vector ABC kit (Vector Laboratories) and 3,3′-diaminobenzidine (DAB; Dojindo, Kumamoto, Japan). For immunofluorescence, sections were incubated with 3% (v/v) normal goat serum and incubated overnight at 4°C with primary antibodies. Appropriate fluorophore-conjugated secondary antibodies (Molecular Probes, Inc., Eugene, OR, USA) were used for visualization.

The primary antibodies used were as follows: rabbit anti-olig2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-NeuN (1:200; Millipore, Bedford, MA, USA), mouse anti-myelin basic protein (MBP) (1:1,000; Covance, Princeton, NJ, USA), adenoma polyposis coli (APC) (CC-1; 1:250; Millipore), rabbit anti-activated caspase 3 (1:200; Millipore), mouse anti-SATBII (1:200; Abcam, Cambridge, UK), rabbit anti-Ctip2 (1:200; Abcam), rabbit anti-Fluoro-Gold (1:3,000; Millipore), mouse anti-parvalbumin (1:1,000; Swant, Bellinzona, Switzerland), mouse anti-calretinin (1:1,000; Swant), and rat anti-somatostatin (1:250; Millipore). The secondary antibodies were as follows: biotinylated anti-rabbit IgG (1:220; Vector Laboratories, Inc.), biotinylated anti-mouse IgG (1:220; Vector Laboratories, Inc), goat anti-mouse Alexa 488 (1:500; Molecular Probes, Inc.), and goat anti-rabbit Alexa 594 (1:500; Molecular Probes, Inc.).

For cell counting, coronal sections (40 μm thick) at the level of the hindlimb-regulating cortex area (bregma: –2.04) were collected. Cells were counted in the motor and sensory areas in each section (400 μm × 400 μm). Following NeuN, Ctip2, SATBII, and GABAergic subtype staining, cells were counted in the motor and sensory areas (400 μm width, I to VI layers). For quantification of MBP immunostaining, MBP signal in the motor and sensory area (I–IV, V–VI layers: 350 μm × 350 μm each) was digitalized at 16 bit using ImageJ software from the National Institutes of Health (NIH, Bethesda, MD, USA). The appropriate intensity value was set as the threshold, and the pixel number above the threshold was considered positive staining. Discrimination of upper (I–IV) and deep (V–VI) layers was determined using Ctip2 (V–VI layer marker) immunostaining. The ratio of ipsilateral to contralateral pixel number is shown in the graphs.

Fluoro-Gold Labeling

At P20, the spinal cord was exposed at the C3–C4 level by laminectomy under anesthesia (n = 5). One microliter of 2% (w/v) Fluoro-Gold (FG; Biotium, Hayward, CA, USA) was injected into the center of the dorsal corticospinal tract (1-mm deep from the dura) with a glass micropipette (tip diameter: 60–80 μm). Rats were anesthetized and perfused with 4% (w/v) PFA 6 days later. Brain sections were cut for histological analysis. FG-labeled cells were detected using an anti-FG antibody. The cell numbers were counted in every eighth brain section in the hindlimb motor-regulating cortex [three sections for each brain, 700 × 700 μm² in motor (M1 and M2) and sensory areas].

Argyrophil-III Silver Staining

Under deep anesthesia with pentobarbital, animals (DWMI: n = 5, sham operated: n = 4) were perfused with 0.1% (w/v) cacodylate (Wako) buffer solution containing 2% (w/v) PFA and 2.5% (w/v) glutaraldehyde (Wako). Brains were removed 24 h after perfusion and postfixed for 1 week at room temperature. Serial coronal sections (40 μm) were cut and processed for argyrophil-III staining. The sections were esterified in 1-propanol (Wako) containing 1.2% (v/v) sulfuric acid (Katayama Chemical., Osaka, Japan) and 2% (v/v) distilled water for 16 h. After rehydration in 50% and 25% (v/v) 1-propanol and distilled water, sections were processed in silicotungstate (Wako) physical developer as described previously^33,34. For cell counting, the number of positive cells in the selected area (400 μm × 400 μm) was counted in the hindlimb-regulating cortex (DWMI 24 h: n = 4, DWMI 48 h: n = 4).

Statistics

Data were analyzed using StatView 5 software (SAS institute, Cary, NC, USA). All data are presented as mean ± SEM, and values of p < 0.05 were considered significant. Student's t-test was used for comparison between sham-operated and DWMI groups in rotarod test. Student's t-test was also used for comparison between contralateral and ipsilateral sides in the number of APC-positive cells, NeuN-positive cells, STABII-positive cells, Ctip2-positive cells, FG-positive cells, and GABAergic markers such as parvalbumin, somatostatin, and calretinin. For the comparison of the percentage of caspase 3-positive cells and NeuN-positive cells, one-way ANOVA followed by Scheffé post hoc test was used in four groups (sham-operated ipsilateral side, sham-operated contrarateral side, DWMI ipsilateral side, DWMI contralateral side). To detect statistical significance in the intensity of MBP staining, one-sample t-test was used for comparison as the intensity of contralateral side was estimated as onefold.

Impaired Motor Coordination and Altered Myelination Were Induced in the DWMI Rat Model

To investigate sensorimotor function in our DWMI rat model, we performed the accelerating rotarod test at adulthood (8 months old). DWMI rats (n = 16) showed a significantly lower score compared with sham-operated animals (n = 12), indicating that motor coordination was impaired (Fig. 1A). In our analysis of motor deficit scores (beam walk ability test, hindlimb retract test, forepaw grasp test) ³⁵ , DWMI rats showed mild functional deficits mainly in the left hindlimb (data not shown). Therefore, we focused on the hindlimb-regulating somatosensory cortex.

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Figure 1.

Coordinated motor dysfunction with myelination failure in the WMI model rat. (A) Motor coordination was assessed using the accelerating rotarod system. The DWMI group showed significantly lower scores compared with the sham group (DWMI: n = 16, sham: n = 12, *p < 0.05 in Student t-test). (B) Immunostaining of APC-positive cells (red) in the ipsilateral white matter with DAPI (blue). Scale bar: 50 μm. Number of APC-positive mature OLs decreased in ipsilateral WM at 6-month-old model rats (n = 4 *p < 0.05 in Student t-test). (C) Weaker MBP immunoreactivity was observed in the M1 and S1 areas of the ipsilateral cortex (2 months old). Scale bar: 100 μm. M1, primary motor cortex; M2, secondary motor cortex; S1, primary sensory cortex. (D, E) Quantification of MBP signal intensity at P26 (n = 5) (D) and P74 (n = 4) (E). Discrimination between upper (I–IV) and deep (V–VI) layers was determined by Ctip2 (V–VI layer marker) immunostaining (*p < 0.05 in one-sample t-test).

Disturbances to OL development after H-I were confirmed in the model by immunostaining to mature OL marker APC. Many cells that expressed APC in the cytoplasm were detected in the WM (Fig. 1B). There was a 16.0% reduction of APC-positive cells in the ipsilateral side of 6-month-old DWMI rats (n = 4) (Fig. 1B). To confirm whether this reduction in mature OLs coincided with a disturbance to myelination, the hindlimb-regulating sensorimotor cortex was immunostained using the MBP antibody. In the ipsilateral cortex, an obvious decline in MBP-positive myelin was observed compared with the contralateral cortex, which extended from the motor area to the sensory area (Fig. 1C). We next quantitated the intensity of MBP signal using ImageJ software in the upper (I–IV) and deep layers (V–VI) of the motor and sensory cortex at P26 (period of active myelination, n = 5) and P74 (period of myelination completion, n = 4). At both developmental stages, MBP intensity significantly decreased in the upper layer of the sensory area (Fig. 1D). There was a slight reduction in MBP signal detected in the upper motor area (P26, p = 0.054; P74, p = 0.07), while no change in MBP intensity was observed in the deep layers of the motor and sensory areas. These data indicate that P3 H-I leads to functional deficits with loss of mature OLs and altered myelination in the upper sensorimotor cortex.

H-I Causes Apoptotic Cell Death of Glial Cells, but not Neurons

To monitor cell death following H-I, we performed active caspase 3 (casp3) immunostaining at 24 and 48 h after injury. Casp3-positive cells were detected in sham-operated control brains (n = 3 for each time points) (Fig. 2A and B) as well as in the motor and sensory cortex of model rats at both time points (24 h: n = 6, 48 h: n = 4). However, no significant difference was observed between H-I and control rats (Fig. 2A and B). Interestingly, the number of casp3-positive cells increased in ipsilateral WM at 24 h compared with the contralateral side (Fig. 2A), indicating that the WM area was sensitive to H-I injury. Because other DWMI animal models have been shown to have more neuronal death after H-I compared to controls, neuronal death was investigated in our model. We monitored the numbers of casp3/NeuN double-labeled cells at 24 and 48 h. No double-labeled neurons were detected at either time point (Fig. 2C). Almost all of the casp3-positive cells were colocalized with olig2 (Fig. 2D) or glial fibrillary acidic protein (GFAP) (Fig. 2E), suggesting that glial cell death mainly occurred in cortical WM. No significant differences in the total number of NeuN-positive neurons were observed in the ipsilateral motor and sensory cortices at 48 h compared with the contralateral cortex (sham: n = 3, DWMI: n = 4) (Fig. 2F).

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Figure 2.

Apoptotic cell death was not evident in neurons after H-I. (A) Immunostaining of active caspase 3 (a-casp3) at 24 h and (B) 48 h after H-I. Although casp3-positive cells were detected in sham-operated control brains (n = 3), no significant difference was detected in the motor and sensory areas at each time point (24 h: n = 6, 48 h: n = 4). Notably, the numbers of positive cells increased in ipsilateral WM at 24 h compared with the contralateral side (A). *p < 0.05 in one-way ANOVA followed by Scheffé F-test. (C, E) Few a-casp3/NeuN double-positive apoptotic neurons were detected at 24 h. Almost all a-casp3-positive cells colocalized with olig2 (D) or GFAP (E). Arrows indicate double-positive cells. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (F) The percentage of NeuN-positive cells in WM and the cortex did not change between DWMI rats (n = 4) and the sham control (n = 3) at 48 h.

As argyrophil-III silver staining is known to detect degenerating neurons at very early phases ³⁴ , we monitored the extent of degeneration using argyrophil-III silver staining at 24 h, 2, 3, and 6 days after injury (DWMI; n = 5, sham: n = 4). In the motor and sensory cortices, H-I did not induce neuronal degeneration (Fig. 3A and B). At 24 h and 48 h after injury, some dark cells with large cell bodies and processes were detected in the deep regions of the ipsilateral cortex (24 h: 13.5 ± 3.2, 48 h: 12.5 ± 3.3, n = 4) [Fig. 3D (inset) and E]. However, these cells were also observed in the contralateral side (24 h: 14.0 ± 4.5 cells, 48 h: 14.0 ± 3.7 cells, n = 4) (Fig. 3E) and sham-operated cortex at the same levels. There was no change in the expression pattern of silver-stained cells (damaged neurons) at days 3 and 6 (data not shown). Thus, apparent neuronal cell loss was not induced after H-I in the sensorimotor cortex in our model.

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Figure 3.

Detection of damaged cells at the very early phase using argyrophil-III staining. (A, B) Argyrophil-III silver staining was carried out at 24 h after H–I (DWMI: n = 5, sham: n = 4). Typical degenerating dark neurons were not detected in either the motor and sensory areas. (C, D) Some positively stained neurons with large cell bodies and processes were detected in the ipsilateral deep cortex (D, inset). However, a similar population of cells was also observed on the contralateral side (C) at 24 and 48 h. Scale bar: 100 μm in (A–D). Inset: Some positive neurons were detected on the contralateral and ipsilateral cortex. Scale bar: 25 μm. (E) Cell counting of argyrophil-III staining positive cells revealed that no significant difference in the number of the positive neurons between the contralateral or ipsilateral cortices at 24 h and 48 h after H–I (24 h: n = 4, 48 h: n = 4).

P3 H-I Injury Does not Affect Cortical and Corticospinal Projections Associated with Hindlimb Motor Function

To investigate whether H-I injury at D3 causes alteration of neural projections in the sensorimotor cortex, we stained cortical projecting cells with anti-SATB II, a marker of CC projecting neurons, and Ctip2, a marker of layers V–VI neurons (Fig. 4A). In the cerebral cortex, SATB II was mainly expressed in layer sII–IV, while some positive staining was detected in deep layers. Ctip2 was detected in layers V–VI (Fig. 4A, magnification). There was no significant difference in the number of SATB II-positive cells in the motor area between the contralateral and ipsilateral side (919 ± 72 cells vs. 955 ± 40 cells, n = 5) (Fig. 4C) as well as in the sensory area (1,082 ± 96 vs. 1,074 ± 66 cells, n = 5) (Fig. 4D). There was also no difference observed between Ctip2-positive cells in the motor area (408 ± 25 cells vs. 371 ± 14 cells, n = 5) (Fig. 4C) and in the sensory area (420 ± 31 vs. 377 ± 33 cells, n = 5) (Fig. 4D). The total number of NeuN-positive neurons in the contralateral and ipsilateral side was almost the same in the motor area (1,187 ± 82 cells vs. 1,170 ± 29 cells, n = 5) (Fig. 4C) and sensory area (1,466 ± 100 vs. 1,422 ± 63 cells, n = 5) (Fig. 4D).

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Figure 4.

Absence of cortical projection neuron loss in the WMI rat model. (A) SATBII-positive cortical projecting neurons (green) and Ctip2-positive layers V–VI neurons (red) were observed in the contralateral and ipsilateral sensorimotor area at P10. Staining revealed that most cells in layers II–IV were SATBII-positive, while Ctip2-positive projecting neurons were detected in layers V–VI. Scale bar: 50 μm. (B–D) Number of SATBII-positive, Ctip2-positive, and NeuN-positive cells in the motor cortex (MC) and sensory cortex (SC) (B). No significant differences in number were observed in the motor (C) and sensory (D) areas between the contralateral and ipsilateral side (n = 4–5).

To estimate the damage to corticospinal neurons in the cortex, the retrograde neuronal tracer FG was injected into the spinal cord at C3–C4 level at P20 and examined 6 days later. FG successfully incorporated into pyramidal neurons and was detected in layer V of the area from the forebrain to the hindbrain (Fig. 5A).

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Figure 5.

H-I does not induce abnormal corticospinal projection. (A, B) Immunostaining of Fluoro-Gold (FG) in the P26 cortex revealed positive cells at layer V in the hindlimb-regulating cortex. Scale bar: 100 μm. The number of FG-positive neurons did not change in either the motor or sensory area at P26 (n = 5) (B). (C, D) Immunostaining of parvalbumin, somatostatin, and calretinin. No significant differences in the number of each subtype were detected in the motor (C) or sensory (D) areas (n = 3–5).

The same number of FG-positive cells was detected in the hindlimb-regulating motor area of the ipsilateral and contralateral sides (245.2 ± 27.9 cells vs. 313.6 ± 26.4 cells, n = 5) (Fig. 5B) as well as in the sensory area (238 ± 27.5 cells vs. 290.6 ± 20 cells, n = 5) (Fig. 5B), indicating that the hindlimb-regulating corticospinal projection was intact after H-I. Furthermore, the number of corticospinal projecting neurons was maintained at 9 months after H-I (data not shown), suggesting that secondary loss of neurons was not induced by disturbances to myelination at the site of injury. As inhibitory neurons have been reported to decrease chronically and delay the development of these inhibitory neurons in a DWMI model^36,37, we monitored the expression of inhibitory interneuron subtypes using antibodies against parvalbumin, somatostatin, and calretinin, (n = 3–5) (Fig. 5C and D). In the P26 rat cortex, no significant change in the number of each cell subtype was detected in the motor (Fig. 5C) and sensory areas (Fig. 5D).

H-I Alters Gene Expression in the White Matter and Sensorimotor Area

To assess the factors that contribute to myelination disturbances and motor dysfunction in our DWMI model, we performed cDNA microarray analysis in the P5 sensorimotor cortex (n = 2 for DWMI and sham operated) (Tables 1 and 2).

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Table 1.

Upregulated Genes in DWMI Rat Model

GO Term	p Value	Gene Symbols
GO:0006508: proteolysis (biological process: BP)	0.000183004	Mmp28, Adamts1, Plau, C1r, Klk4, Klkb1, Acr, Capn8, Ace
GO:0005975: carbohydrate metabolic process (BP)	0.000178457	Hk3, Igf2, Galm, Kl, Pofut, Slc3a1
GO:0019233: sensory perception of pain (BP)	0.000253183	Aqp1, Ednra, Ace, Cnr2
GO:0006874: cellular calcium ion homeostasis (BP)	0.000235407	Fxyd1, Slc37a4, Ccl2, Trpv4
GO:0001666: response to hypoxia (BP)	0.000347279	Ednra, Plau, Sod3, Tf, Ccl2, Ace
GO:0014070: response to organic cyclic compound (BP)	0.00032326	Ednra, Igf2, Slc37a4, Tf, Fads1, Fos
GO:0050729: positive regulation of inflammatory response (BP)	0.000453131	Ednra, Fabp4, Ace
GO:0007568: aging (BP)	0.000533321	Ednra, Kl, Fads1, Ccl2, Fos
GO:0006811: ion transport (BP)	0.000828529	Fxyd1, Slc39a9, Tf, Kcnj13, Kcne2, Chrna6, Trpv4
GO:0032496: response to lipopolysaccharide (BP)	0.000815962	Ednra, Ccl2, Ace, Fos, Cnr2
GO:0042493: response to drug (BP)	0.00085534	Aqp1, Fabp4, Igf2, Slc1a3, Ccl2, Htr2c, Fos
GO:0008217: regulation of blood pressure (BP)	0.00119879	Ednra, Sult1a1, Ace
GO:0006950: response to stress (BP)	0.00182874	Sod3, Sult1a1, Fads1, Fos
GO:0010951: negative regulation of endopeptidase activity (BP)	0.00186812	Serpinc1, Serpinb1a, Renbp
GO:0043066: negative regulation of apoptosis (BP)	0.00212697	Aqp1, Ednra, Msx1, LOC303341, Hspb1
GO:0006470: protein dephosphorylation (BP)	0.00437192	Ppm1b, Styxl1, Eya2
GO:0001822: kidney development (BP)	0.00478655	Adamts1, Sulf1, Ace
GO:0007584: response to nutrient (BP)	0.00846179	Serpinc1, Slc37a4, Fads1
GO:0051384: response to glucocorticoid stimulus (BP)	0.00991436	Fabp4, Sult1a1, Ccl2
GO:0008152: metabolic process (BP)	0.0107682	Mmp28, Adamts1, Sulf1, Sult1a1, Acr
GO:0006954: inflammatory response (BP)	0.0133784	Itgb6, Klkb1, Ccl2
GO:0006813: potassium ion transport (BP)	0.0145472	Aqp1, Kcnj13, Kcne2
GO:0006810: transport (BP)	0.0143977	Fads1, Slc3a1, Ramp3, Ttr
GO:0007507: heart development (BP)	0.0185241	Ednra, Msx1, Pofut1
GO:0055114: oxidation-reduction process (BP)	0.0494169	Degs2, Sod3, Fads1

Mmp28, matrix metallopeptidase 28; Adamts1, a disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 1; Plau, plasminogen activator; C1r, C1r protein; Klk4, kallikrein-related peptidase 4; Klkb1, kallikrein B, plasma 1; Acr, acrosin prepropeptide; Capn8, calpain 8; Ace, angiotensin I-converting enzyme 1; Hk3, hexokinase 3; Igf2, insulin-like growth factor 2; Galm, galactose mutarotase; Kl, klotho; Pofut1, protein O-fucosyltransferase 1; Slc3a1, solute carrier family 3, member 1; Aqp1, aquaporin 1; Ednra, endothelin receptor type A; Cnr2, cannabinoid receptor 2; Fxyd1, FXYD domain-containing ion transport regulator 1; Slc37a4, solute carrier family 37 (glucose-6-phosphate transporter), member 4; Ccl2, chemokine ligand 2; Trpv4, transient receptor potential cation channel, subfamily V, member 4; Sod3, superoxide dismutase 3, extracellular; Tf, Serotransferrin precursor; Fads1, fatty acid desaturase 1; Fos, FBJ osteosarcoma oncogene; Fabp4, fatty acid-binding protein 4 adipocyte; Slc39a9, solute carrier family 39 (zinc transporter), member 9; Kcnj13, potassium inwardly rectifying channel, subfamily J, member 13; Kcne2, potassium voltage-gated channel, Isk-related subfamily, gene 2; Chrna6, cholinergic receptor, nicotinic, a polypeptide 6; Slc1a3, solute carrier family 1, member 3; Htr2c, 5-hydroxytryptamine receptor 2C; Sult1a1, sulfotransferase family 1A, phenol-preferring, member 1; Serpinc1; serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1; Serpinb1a, serine (or cysteine) peptidase inhibitor, clade B, member 1a; Renbp, rennin-binding protein; Msx1, homeobox, msh-like 1; Hspb1, heat shock protein 1; Ppm1b, protein phosphatase 1B, magnesium dependent, β isoform; Styxl1, serine/threonine/tyrosine interacting-like 1; Eya2, eyes absent 2 homolog; Sulf1, sulfatase 1; Serpinc1, serine (or cysteine) peptidase inhibitor, clade C (antithrombin), member 1; Fabp4, fatty acid-binding protein 4, adipocyte; Itgb6, integrin β 6; Klkb1, kallikrein B, plasma 1; Slc3a1, solute carrier family 3, member 1; Ramp3, receptor (calcitonin) activity-modifying protein 3; Ttr, transthyretin gene; Degs2, degenerative spermatocyte homolog 2 (Drosophila), lipid desaturase; Sod3, superoxide dismutase 3, extracellular.

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Table 2.

Downregulated Genes in DWMI Rat Model

GO Term	p Value	Gene Symbols
GO:0006811: ion transport (BP)	0.000111839	Kcnj16, Cacna2d2, Hcn2, Gabrg3, Chrnb4, Clic5, Accn4
GO:0006955: immune response (BP)	0.00319535	Il2rg, Il7, RT1-S3, Oas1b
GO:0007186: G-protein-coupled receptor protein-signaling pathway (BP)	0.00804821	Olr121, Taar1, Olr1507, Olr482, Olr1375, Olr491, Olr192, Olr434, Olr1350

Kcnj16, potassium inwardly rectifying channel, subfamily J, member 16; Cacna2d2, calcium channel, voltage-dependent, α 2/δ subunit 2; Hcn2, hyperpolarization-activated, cyclic nucleotide-gated K+ 2; Gabrg3, γ-aminobutyric acid (GABA) A receptor, subunit γ 3; Chrnb4, cholinergic receptor, nicotinic, β polypeptide 4; Clic5, chloride intracellular channel 5; Accn4, amiloride-sensitive cation channel 4, pituitary; Il2rg, interleukin 2 receptor, γ chain; Il7, interleukin 7; RT1-S3, RT1-149 protein; Oas1b, 2′-5′ oligoadenylate synthetase 1B; Olr121, olfactory receptor Olr121; Taar1, trace amine-associated receptor 1; Olr1507, olfactory receptor Olr1507; Olr482, olfactory receptor Olr482; Olr1375, olfactory receptor Olr1375; Olr491, olfactory receptor Olr491; Olr192, olfactory receptor Olr192; Olr434, olfactory receptor Olr434; Olr1350, olfactory receptor Olr1350.

Of the 163 genes that changed expression, 98 were upregulated, and 65 were downregulated in the ipsilateral sensory motor cortex compared with the contralateral cortex. Gene ontology (GO) analysis using GENE CODIS2.0 (http://genecodis2.dacya.uccm.es/) revealed that genes involved in the carbohydrate metabolic process, proteolysis, ion transport, negative apoptosis regulators, hypoxia-responsive factors, inflammatory response, and oxidation–reduction process were upregulated in the ipsilateral cortex (Table 1). Among the downregulated genes, many were related to ion transport and the G-protein-coupled receptor protein signaling pathway (Table 2). Many genes responsible for neuronal survival, myelin loss, and differentiation were also altered in the DWMI model.

Specific Feature of the DWMI Rat Model

The significant feature of our DWMI rat model was minor neuron damage compared with previous reports^29,38,39. Distinct cell death of neurons was not shown in the sensorimotor cortex after H-I; however, CC1-positive mature OLs were decreased at adulthood. As described by previous reports, Fluoro-Jade (FJ) B-positive degenerating neurons were detected in deep layers (layer VI) ³⁸ , an area vulnerable to neonatal H-I^39,40. We also detected FJ B-positive cells at 24 and 48 h in deep layers (layers IV–V) in the DWMI model (data not shown). In contrast, no neuron damage was detected when we used the argyophil-III staining method to detect the very early phase of neuron damage ⁴¹ . Assessment of cell death using FG injection, STABII, Ctip2, and GABAergic marker labeling strongly confirmed no neuronal loss in the somatosensory cortex in the DWMI model. In addition, few casp3/NeuN double-positive apoptotic neurons were detected in the cortex at 24–48 h after the lesion. These data indicate that neuron damage in the somatosensory cortex of DWMI rats was minor following H-I and not aggressive enough to induce significant cell death.

The principal target in the acute phase of WMI is O4-positive pre-OLs because they are selectively vulnerable to H-I^{15,18–22,42}. Three classes of MRI-defined WMI at the chronic phase have also recently been revealed in a sheep model of WMI: DWMI, focal necrotic WMI, and microscopic necrotic WMI^3,14. In particular, DWMI is a major form of chronic WMI and is characterized by astrogliosis and myelination disturbances. Using histological assessment, casp3-positive apoptotic cells only increased in ipsilateral WM at 24 h after H-I (Fig. 2A and B), which were mostly olig2 and casp3 double-positive OL lineage cells and some reactive GFAP-positive cells. Reduced MBP staining in layers I–IV of the sensory cortex was revealed at 6 months (chronic phase) compared with the contralateral cortex. Furthermore, a small, but significant, reduction in CC1-positive mature OLs was detected in WM, while a transient increase in OPCs was observed (data not shown). These data suggest that both the cell death of OL lineage cells at the acute phase and OPC maturation arrest at the chronic phase may contribute to myelination failure in the upper cortical layers in adulthood. We also suggest that this WMI rat model may be an appropriate DWMI model as it depicts the major form of chronic WMI, including myelination disturbances in OL lineage cells and reactive responses in astrocytes.

Transient reduction of excitatory neurons in the cerebral cortex and prolonged decreases, or developmental delays, to inhibitory interneurons was reported in chronic hypoxia from P3 to P11^36,37,43. In our DWMI model, no expression of casp3 was detected in NeuN-positive neurons at 24–48 h after H-I (Fig. 2A and B), and no reduction in the total number of neurons was observed in the P10 cortex (Fig. 2D). Notably, no decrease in the number of both excitatory neurons (retrogradely labeled FG-positive layer V cells) and inhibitory neurons (parvalbumin-, somatostatin-, calretinin-positive cells) was observed in the DWMI rat model following H-I at P3. Thus, neurons in the somatosensory cortex were not damaged by H-I in the DWMI rat model rather than the recovery after transient reduction or delays such as in chronic hypoxia. With respect to differences between our DWMI model and previously reported animal models, it is necessary to consider the differences in pathology caused by the experimental conditions such as the age of the animal and the duration of H-I.

Probable Mechanism for Functional Deficits

Although no neuronal loss in the sensorimotor cortex was observed, dysfunction to sensory/motor coordination was revealed using the accelerating rotarod test. One possibility for this functional deficit could be a disturbance to neuron–glia interaction as the intensity of myelin staining was lower in the upper layer in the DWMI brain (myelination failure). For example, myelin loss may change conduction velocity in the sensorimotor inputs–outputs and/or intracortical association, which leads to an imbalance in information processing for coordinated movement. It is possible that synaptic input from the hypothalamus and/or intracortical interaction between layers I–IV dendrites was affected in the DWMI model, resulting in dysfunction to sensory motor coordination.

Another possible reason for the functional deficits may be neuronal reorganization in the cortex. Prenatal ischemia was reported to induce somatosensory map disorganization and neuronal reduction in adulthood ⁴⁴ . Although we did not detect cell loss in CC projecting neurons, V–VI layer cortical neurons, or corticospinal projection neurons, it is possible that drastic changes in connectivity between areas or cortical map reorganization in motor and somatosensory areas occurred, which could modulate sensorimotor function accompanied with myelination failure. In our preliminary data, cortical map changes following intracerebral microstimulation was shown in the DWMI model brain.

We focused on the neuronal damage responsible for hindlimb sensorimotor function. Several possible reasons are considered in altered dysfunction especially in the hindlimb sensorimotor cortex; further investigations are required to understand the mechanism of altered dysfunction after H-I in the future.

In our preliminary data, the deficit of motor coordination was shown as early as P28 accompanied with myelination failure (Fig. 1E). Similarly, hindlimb motor dysfunction in beam walk ability test and hindlimb retract test was also observed at 2 months (data not shown). Thus, it is noteworthy that a correlation of behavioral impairment with histological damage in myelination was shown in both P26 and P74 (Fig. 1D and E).

Although cystic PVL cases show gray matter damage in some brain areas, including the cerebral cortex, noncystic PVL including DWMI showed less neuronal loss ¹² . From our analysis, the DWMI rat model may replicate more diffuse and mild WMI cases without necrosis. This model is useful for pathological analysis of DWMI motor dysfunction induced mainly by myelin deficits without obvious neuronal loss.

Cell-Based Therapies Using the DWMI Model

In the DWMI model that predominantly involves pre-OL degeneration followed by myelination failure, OL lineage cells such as pre-OLs will be an appropriate donor cell type. Embryonic stem cell-differentiated OL lineage cells are suggested to be the preferred cell type for transplantation in the treatment of WMI ²⁶ . The neuroprotective effect of trophic support from grafted OL progenitor cells has recently been reported in a rat PVL model ²⁴ . Therefore, it is very likely that OL lineage cells derived from iPSCs ²⁷ may be an appropriate donor cell type for recovery of motor dysfunction following DWMI.

Regional differences in OL differentiation in the adult brain revealed the role of gray matter and WM niches using homo- and heterotopic transplantation ²³ . A preference of O2A progenitors to differentiate into OLs rather than astrocytes was observed in the neonatal rat brain ⁴⁵ . In this study, cDNA microarray in the P5 sensorimotor cortex revealed 98 upregulated and 65 downregulated genes in the ipsilateral sensory motor cortex compared with the contralateral cortex (Tables 1 and 2). OPC-related genes such as PDGFR-α, NG2, and olig2 were not included in the 163 altered genes, indicating that intrinsic OPCs lost proliferative capacity in gene expression at P5. The gene expression of MBP and PLP myelin protein was not altered by H-I; presumably it is too early to be expressed at this time point. Interestingly, some genes were associated with cell survival, myelin loss, and differentiation of OL lineage cells. Thus, it is possible that replacing lost cells with cells at different stages of maturation (OL lineage cells vs. pre-OLs) has a greater effect than exposure to neurotrophic substances.

Immune responses such as inflammation after H-I and rejection after cell transplantation should be taken into consideration for cell transplantation research. As the neonate has high ability to generate CD4⁺Foxp3⁺ regulatory T cells (Tregs) ⁴⁶ and this ability gradually becomes weaker within 2 weeks after birth, it might be better for us to graft OL lineage cells at earlier stage (P3–P5) after birth when OPCs are proliferating, and the possibility of rejection is less likely. Thus, our DWMI model would be an appropriate model to minimize the rejection after cell transplantation. Future studies should aim to assess OL transplant route, dose, and timing in relation to WMI, OL lineage cell grafts for DWMI, and survival and differentiation of OL lineage cells in vivo. In addition, ways to improve myelination failure of pre-OLs and functional recovery after engraftment need to be addressed.