Male Sprague-Dawley rats, 8- to 9-week-old and weighing 200–240 g, were housed two per cage in a controlled husbandry unit with a 12/12 hour light/dark cycle and permitted ad libitum access to food and water. The temperature was maintained at 21 ± 1˚C. A total of 77 rats were used for the experimental study, and they were divided into three groups: 50 rats for light microscopic histological examination, 13 rats for electron microscopic examination, and 14 rats for measurement of infarct volume. The brain was harvested from 3 hours to 21 days after the photothrmobotic ischemia and infarct volumes were measured at 14 days (Table 1). Animal experiments were performed according to the institutional guidelines of Chonnam National University Research Institute of Medical Science and Gwangju Institute of Science and Technology (GIST). All efforts were made to minimize animal suffering and to reduce the number of animals used. Institutional ethical committee approval was obtained prior to the experiments.

The photothrombotic capsular infarction model used in this study was described previously [21]. Experimental rats were divided into two groups: stroke lesion group (SLG) and sham-operated group (SOG). Briefly, experimental rats of SLG were anesthetized and fixed with a small animal stereotactic frame. A 28-gauge straight stainless steel needle implemented with a fine optical fiber transmitting laser light, a 125 μm outer diameter, and a 62.5 μm core diameter, was inserted through a midline scalp incision. The needle was stereotactically inserted into the PLIC target; 2.0 mm posterior, 3.1 mm lateral to midline, and 7.2 mm deep to bregma, as shown in Fig. 1. The coordinate used in this experiment was deeper than those previously suggested [16]. Rose Bengal dye was injected through the tail vein, followed by 1.5 minutes of laser irradiation with an intensity of 2.5 mW at the fiber tip. After the scalp wound was secured, the rat was transferred to a recovery chamber. The experimental rats of SOG underwent an identical lesion-making procedure except for receiving an injection of saline solution (0.2 mL/100 g) instead of Rose Bengal dye.

The behavioral tests employed were described in the previous study [21], and single pellet reaching tasks (SPRTs) were used to measure motor performance in this study. Briefly, an experimental apparatus was made of clear Plexiglas with a 1-cm wide slit in the middle of the front wall for the food shelf. Once rats began to show a forelimb preference to obtain a sucrose pellet (Bio-Serve, Frenchtown, NJ, USA) through the slit, a pellet was obliquely placed contralateral to the preferred paw to prevent the use of the nonpreferred paw. Animals were subsequently administered 20 pellets per session for 21 days. Successful reach was defined as a reach in which an animal grasped a food pellet, brought it into the cage, and consumed it without dropping. Reaching performance was scored and the success rate was calculated as the formula, number of successful reaches × 100/20 scores before the operation were averaged to represent preoperative reaching performance. After the photothrombotic infarct, the experimental rats were subdivided as follows from the SPRT score: early recovery group (ERG) characterized by > 50% recovery in 7 days; moderate recovery group (MRG) characterized by > 50% recovery of the prelesion status in 14 days; and poor recovery group (PRG) which had reaching performance at < 50% of the prelesion status. Subjects in the ERG were excluded from the study. Persistent motor impairment, characteristic of both MRG and PRG subjects, developed effectively in 71% of rats. SOG rats did not show a significant decrease in SPRT performance within the observation period.

To measure the infarct size in both MRG and PRG rats, seven fresh brains were perfused at 14 days after the ischemia. Coronal brain sections of 2.5-mm thickness were obtained from the center of the needle insertion sites using a metallic tissue plate covered with optimal cutting temperature (OCT) compound and immediately frozen at –25˚C in a frozen tissue microtome. Serial tissue slices from the frozen brains (20-µm thick sections at 200-µm intervals) were prepared and stained with hematoxylin and eosin (H&E) and cresyl violet. Histologic images were scanned with a ScanScope virtual microscope (Aperio Technologies, Vista, CA, USA). Images from five to six sections were assessed for infarct lesions with Image J software (NIH, Bethesda, MD, USA). The total volume of brain injury was determined by summing the area of damaged tissue recorded from all sections and multiplying by the distance between sections [23]. The completeness of the IC destruction was determined by three of the authors, all of whom were blinded to the motor recovery group.

Experimental time-points after PLIC ischemia were as follows. To assess early changes of PLIC infarct, n = 2 each at 3, 6, and 12 hours, and n = 4 each at 1, 4 and 7 days. To assess late changes between MRG and PRG infarcts, n = 8 each at 14 and 21 days. For SOG, n = 2 for each postlesion time-points. After the behavioral evaluation, rats were anesthetized with intraperitoneal sodium pentobarbital (70 mg/kg) and killed by intra-aortic perfusion of 1% paraformaldehyde in phosphate buffer solution (PBS; 0.12 M, pH 7.4) delivered by a perfusion pump (36 mL/min) for 5 minutes followed by 4% paraformaldehyde in PBS for 15 minutes at the same flow rate. The brains were then removed and coronally sectioned into 4-mm-thick slices surrounding the center of the needle insertion site. For light microscopy examinations, the brains were removed from the skull, postfixed in 4% paraformaldehyde for 2 days at room temperature, and subsequently kept in processed paraffin-embedded tissue blocks. Routine H&E, luxol fast blue–periodic acid-Schiff (LBPAS), and immunohistochemical stainings were performed on tissue sections (6-µm-thick) on probed-glass slides. Immunohistochemical staining with anti–glial fibrillary acidic protein (GFAP; 1:400, polyclonal, Zymed, South San Francisco, CA, USA) and anti–neurofilament protein (NFP; 1:600, clone 2F11, Dako Cytomation, Carpinteria, CA, USA) was done using the avidinbiotin peroxidase method [24] with the ABC kit (Vector Laboratories, Burlingame, CA, USA). Antigen retrieval was conducted by heating the tissue sections at 60˚C for 5 minutes in sodium citrate buffer, after which the sections were incubated in a mixture of 3% hydrogen peroxide and 10% methanol for 20 minutes, followed by 10% goat serum for 1 hour at room temperature. The sections were incubated with each primary antibody for 2 hours, biotinylated secondary antibody for 20 minutes, and streptavidin-horseradish peroxidase (Dako Cytomation) for 20 minutes. They were then developed with a chromogenic solution of diaminobenzidine and counterstained with hematoxylin to visualize cell nuclei. Known positive and negative tissues were used as controls.

One rat each at 3, 6, and 12 hours, and two rats each at 1, 4, 7, 14, and 21 days after the ischemia (n = 13) and one SOG rat were studied by transthoracic cardiac perfusion [25]. Rats were perfused with 1% paraformaldehyde/0.1% glutaraldehyde in cold phosphate buffer (PB) delivered by a perfusion pump (36 mL/min) for 5 minutes, followed by 2% paraformaldehyde/2% glutaraldehyde in cold PB at the same flow rate for 5 minutes. Brains were removed and coronally cut into 2-mm-thick slices. After the lesion was evaluated macroscopically in the brain slices, tissues were trimmed to approximately 2 × 2 × 1 mm, placed in 4% glutaraldehyde in PB for 24 hours at 4˚C, postfixed in 2% osmium tetroxide for 2 hours at room temperature, rinsed in 0.1 mol/L cacodylate buffer, dehydrated in graded concentrations of ethanol followed by propylene oxide, incubated in a mixture of epon/propylene oxide (50/50, then 80/20 for 4 hours), and embedded in epon at 60˚C in an oven for 2 days. Semi-thin sections (1-μm-thick) were stained with 1% toluidine blue and screened by light microscopy to select thin section areas from multiple blocks at each lesion time-point for electron microscopy. Selected thin sections (800-Å thick) were cut with a diamond knife on an ultramicrotome (LKB-Produkter, Bromma, Sweden), mounted on copper grids, contrasted with uranyl acetate and lead citrate, and viewed on a JEOL 120S electron microscope (Nihon Denshi, Tokyo, Japan). The investigators were initially blinded to the treatment conditions. Axon and myelin thicknesses were quantified from 3 hours to 21 days after ischemia. Two random fields per grid from two grids were analyzed per experimental brain. Four random images within each field were collected at × 5,000 magnification and analyzed with ImagePro Plus (MediaCybernetics, Rockville, MD, USA). The axon and myelin sheath diameters were determined for every myelinated axon from a minimum of 30 axons for each experimental group. The myelin thickness was calculated by subtracting the axon diameter from the total fiber diameter. The g-ratio of each myelinated axon was calculated by dividing the average axon diameter by the average total fiber diameter [26].

Data were analyzed with SPSS ver. 10.0 statistical analysis software (SPSS Inc., Chicago, IL, USA). SPRTs for skilled reaching performances were analyzed with a repeated measure analysis of variances for group and time. Electron microscopy data (g-ratio, axon caliber, and myelin thickness) were tested with a one-way ANOVA. Data are presented as the mean ± standard error of mean, with p < .05 considered significant.

A series of frozen tissue slices stained with cresyl violet to measure infarct volume is illustrated in Fig. 2A. Infarct volume included the area of necrosis and surrounding demyelination and astrogliosis. At 14 days after ischemia, the infarct volume was 0.6 ± 0.2 mm³ in the PRG and 0.5 ± 0.2 mm³ in the MRG (Fig. 2B). Infarcts in the PRG were slightly larger than those in the MRG; however, there was no significant relationship between infarct volume and the degree of motor recovery in each group.

Myelinated axons in the PLIC were well defined by LBPAS and NFP immunohistochemical staining. The presence of reactive astrocytes was clearly indicated by GFAP immunohistochemical staining. At 3 hours post-ischemia, a pale focal lesion was observed on H&E stained preparation (Fig. 3A). The lesion boundary was not clear and gradually merged with the neighboring normal WM. The lesion was highlighted by light brown positivity on GFAP staining (Fig. 3B). Focal lesions of myelin pallor showed separated, swollen myelinated axons in edematous PLIC by LBPAS staining. Nuclear pyknosis of oligodendrocytes was identified in the lesion. Edematous change was noted in the upper portion of the optic tract and was limited to the vicinity of PLIC.

At 6 hours to 1 day poststroke, the focal lesion of PLIC was more clearly identifiable with time through low magnification microscopy. A focal infarct manifested as a well-demarcated, pale, whitish lesion due to the lack of staining with H&E and LBPAS (Fig. 3C, D). Immunohistochemical staining revealed increased positivity for GFAP (Fig. 3E) and decreased positivity for NFP (Fig. 3F). Immunopositivity for GFAP in the infarct lesion indicated the reactive change of astrocytes. No inflammatory cell infiltration in the infarct lesion was noted.

At 4 days post-ischemia, myelinated axons in the PLIC were completely lost by LBPAS staining (Fig. 3G). The lesion had a central geographic necrosis surrounded by a wall of macrophages, in which each macrophage had a central nucleus with prominent nucleoli and abundant cytoplasm phagocytized with light-blue or reddish granular debris (Fig. 3H). Loss of NFP staining in the PLIC also indicated lesion developed. Neutrophil did not infiltrate in the lesion. Strong GFAP staining highlighted presence of hypertrophic astrocytes along the periphery of the infarct lesion and neighboring WM (Fig. 3I).

At 7 days post-ischemia, the necrotic center showed a decreased volume, and was surrounded by infiltrating macrophages, reactive hypertrophic astrocytes, and newly formed capillaries. Infiltrating macrophages actively phagocytized reddish granular debris throughout the infarct lesion, clearly shown by LBPAS staining (Fig. 3J). At 14 to 21 days post-ischemia, central necrosis was distinctly resolved and the lesion was a pseudocystic with loosely arranged cellular elements. The lesion consisted of a mixture of foamy macrophages, hypertrophic astrocytes with elongated processes, and newly formed capillaries. Critical histopathological features related to motor dysfunction differences between the MRG and PRG were investigated. A portion of myelinated axon bundles passing the PLIC was preserved, as illustrated by immunohistochemical stains for GFAP and NFP and LBPAS staining (Fig. 3K, L). However, the fiber tract of myelinated axons forming the PLIC was severely disrupted and formed pseudocysts in the PRG group, which was clearly identified by LBPAS staining at low magnification (Fig. 3M). In the interpretation of PLIC injury by light microscopy, careful examination of serial tissue sections is important. At 21 days post-ischemia, the focal infarct lesion persisted as a pseudocystic cavity. A few macrophages were still observed along the inner side of the angiogliotic cavity (Fig. 3N). Microscopic examinations confirmed the presence of PLIC lesions (Table 2). The optic tract adjacent to the IC was partially injured in a few rats. However, the adjacent striatum and anterior limb of the IC were intact. The SOG did not show any damage within the underlying IC.

Histopathological alterations resulting from early ischemic injury were further studied by electron microscopy (Table 2). In SOG, normal myelinated axons presented as a unit of a compact lamellar sheath closely encompassing an axon, and oligodendrocytes were surrounded by myelinated axons (Fig. 4A). Size measurement revealed two types of myelinated axons: large (0.85 ± 0.18 µm diameter) and small (0.41 ± 0.13 µm). Even large axons presented in groups or singular aligned intimately with small axons. Each axon was filled with organized intermediate filament structures; called neurofilaments, and mitochondria were occasionally identified within axons. Progressive structural abnormalities in myelinated axons were observed within the ischemic lesion.

At 3 hours post-ischemia, platelets aggregated in the capillary lumen and exudative changes were present in interstitial spaces among myelinated axons. Nodular swelling with loosening of the myelin sheath and irregular wrinkling of axons was observed in both large and small myelinated fibers (Fig. 4B). Nodular loosening of the myelin sheath led to the formation of lamellar knob. With nodular loosening of myelin, a clear space developed in the submyelin area and contacted wrinkled axons with polygonal outlines. However, axonal integrity consisting of filamentous structures and mitochondria was relatively well preserved. Oligodendrocytes had relatively intact nuclei, rough endoplasmic reticulum (ER), and mitochondria. Myelin pallor observed by light microscopy in the early stage of the ischemic injury matched the ultrastructural edematous change in the myelin sheath, submyelin space, and wrinkled axons.

At 6 hours post-ischemia, whorled myelinated axons were formed by progressive loosening of the compact myelin lamellae covered with wrinkled axons (Fig. 4C). Large and small axons identified in SOG rats did not distinct significantly in MRG and PRG groups due to irregular loosening of the myelin sheath and markedly swollen axons. Small hydropic vacuoles developed, and intermediate filaments dissolved in wrinkled axons. At 12 hours to 1 day post-ischemia, the capillary lumen was obliterated by fibrin thrombosis. Nodular loosening of the myelin sheath decreased markedly, and wrinkled axons changed to plump, swollen axons. Swollen axons were filled with many mitochondria, vesicles, and granules, and showed mildly increased electron density (Fig. 4D). With rupture of the myelin sheath,

axonal organelles (mitochondria, neurofilaments, and vesicles) extruded. Large vacuoles formed by demyelination with axonal flooding were common. Expansion of the extracellular space containing edema fluid presented. Reactive astrocytes were identified by clubbed hypertrophic processes containing abundant intermediate filaments that intimately connected with swollen axons (Fig. 4E). Oligodendrocytes had variable pathologic features ranging from intact nuclei and mildly swollen ER in the cytoplasm to peripheral clumping of coarse chromatin along the nuclear membrane. Oligodendrocytes undergoing apoptosis in the ischemic lesion were identified by irregular clumping of coarse nuclear chromatin at the periphery of the nucleus, detachment of the nuclear membrane, and an electrondense cytoplasm filled with swollen mitochondria and ER (Fig. 4F).

At 4 days post-ischemia, whorled myelinated axons were present though the pattern differed from those at 6 hours. The whorls were formed by swollen axons filled with cell organelles and electrondense fine filamentous materials covered with whorled lamellae of myelin sheaths (Fig. 4G). Demyelinated ghost axons packed with edematous flooding were common. Macrophages had phagocytized degenerated and demyelinated axons and electrondense bodies (Fig. 4H).

At 7 to 21 days post-ischemia, the exudative reaction subsided progressively. A number of unmyelinated or thinly myelinated axons were evident. Variably degenerated myelinated axons with an infiltration of scavenging macrophages were seen in the presence of hypertrophic astrocytes and oligodendrocytes. The ultrastructural features were compatible with a pseudocystic wall observed by light microscopy. The number of macrophages decreased with time, but hypertrophic astrocytes were still evident at 21 days post-ischemia.

Axon and myelin thickness was further separately assessed to identify their contributions to g-ratio changes. Axon diameter significantly decreased (p < .001) at 3 hours to 6 hours post-ischemia. However, myelin thickness (calculated by nerve fiber diameter minus axon diameter) remarkably increased (p < .001) at 3 to 6 hours, and then decreased at 24 hours post-ischemia. The increased axon diameter and decreased myelin thickness at 4 to 21 days post-ischemia contributed to an increased g-ratio in the experimental animals. G-ratios did not differ between sham subjects and the contralateral PLIC in the experimental group.