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Introduction

Transient cerebral ischemia leads to selective neuronal damage/death in certain brain regions, including the hippocampal CA1 region (1,2). In the hippocampal CA1 region, transient cerebral ischemia-induced neuronal death occurs in pyramidal neurons several days after ischemia-reperfusion, and this phenomenon is referred to as ‘delayed neuronal death’ (1). However, the underlying mechanisms that are associated with ischemia-induced delayed neuronal death have not yet been fully elucidated (3–5). Following the onset of transient cerebral ischemia, dysfunction of cerebral microcirculation occurs, including cerebral blood-brain barrier (BBB) breakdown and increase in BBB permeability (6–10), resulting in the extravasation of serum proteins in the perivascular space and brain parenchyma (6,10,11). The dysfunction of cerebral microcirculation following ischemic insult is known to be closely associated with the ischemia-induced neuro-inflammation, cerebral edema and neuronal injury (12).

Albumin, the most abundant plasma protein, has multifunctional properties, including maintaining the osmotic pressure of plasma and specifically binding to low molecular weight molecules (13). Several studies have reported that treatment with albumin improves neurological score and decreases histopathological damage in experimental animal models of focal and global cerebral ischemia (14–17). These neuroprotective effects of albumin are thought to be due to its properties, such as its antioxidant activity, its inhibitory activity on pathological platelet aggregation, and its role in regulation of endothelial integrity (18,19). However, few studies regarding albumin expression and its change in neurons or glial cells in the hippocampal CA1 region following transient cerebral ischemia exist to date. Therefore, in the present study, albumin expression in the hippocampal CA1 region of the gerbil, a popular animal model of transient cerebral ischemia (20), was examined following 5 min of transient cerebral ischemia. This suggests that albumin has the potential to be an attractive target for the treatment of cerebral ischemia.

Materials and methods

Experimental animals and induction of transient cerebral ischemia

Male, 6-month old Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center of the Kangwon National University (Chuncheon, Republic of Korea) and were housed according to the animal handling and care guidelines as in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th edition, 2011) (21). The present study was approved by the Institutional Animal Care and Use Committee of Kangwon National University (Chuncheon, Korea; approval no. KW-130424-1).

The surgical procedure for transient cerebral ischemia was performed as previously described (22). In brief, the animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. The complete interruption of blood flow mediated by the 5 min occlusion of bilateral common carotid arteries was confirmed by observing the central artery in retinae under an ophthalmoscope. Normothermic (37±0.5°C) conditions were maintained prior to, during and following the surgery until the animals completely recovered from anesthesia. Sham-operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded.

Cresyl violet (CV) staining and immunohistochemistry for albumin

Sections were prepared from the sham and ischemia-operated animals (n=7 at each time-point) at 2 and 4 days after reperfusion, according to the previously published procedure (22). In brief, the animals were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brain tissues were then serially sectioned into 30 µm coronal sections and they were then placed into 6-well plates containing phosphate-buffered saline.

To examine cellular distribution and damage CV staining was performed as previously described (23). In brief, CV acetate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved at 1.0% (w/v), and glacial acetic acid was added at 0.28% in this solution. The sections were stained and dehydrated by immersing in serial ethanol baths.

Albumin immunohistochemistry was carried out according to our previously published procedure (22). Briefly, sections were incubated with mouse anti-albumin (catalog no. ab19194; dilution 1:100; Abcam, Cambridge, MA, USA), exposed to biotinylated goat anti-mouse immunoglobulin (Ig) G and streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA, USA) and visualized with 3,3′-diaminobenzidine. In order to confirm the cell type containing the albumin immunoreactivity, sections were also processed by double immunofluorescence staining. In brief, sections were incubated in a mixture of mouse anti-albumin (dilution 1:50; Abcam) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; an established microglia marker; catalog no. 019–19741; dilution 1:100; Wako Pure Chemical Industries, Ltd., Osaka, Japan), followed by a mixture of Cy3-conjugated goat anti-mouse IgG (dilution 1:200) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (dilution 1:200) (both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The immunoreactions were observed under a confocal microscope (LSM 510 META NLO; Carl Zeiss AG, Oberkochen, Germany).

For the analysis of albumin immunoreactivity, six sections per animal in each group (n=7) were selected with 120 µm intervals to quantitatively analyze albumin immunoreactivity. Digital images of the hippocampal CA1 region were captured with an AxioM1 light microscope (Carl Zeiss AG) equipped with a digital camera (Axiocam; Carl Zeiss AG). According to our previously published procedure (22), semi-quantification of the immunostaining intensities was evaluated with digital image analysis software (MetaMorph 4.01; Universal Imaging, Inc., Bedford Hills, NY, USA). The mean intensity of the albumin immunostaining in each immunoreactive structure was measured using a 0–255 gray scale system. The level of immunoreactivity was scored as follows: -, no staining (gray scale value: ≥200) ±, weakly positive (gray scale value: 150–199); +, moderate (gray scale value: 100–149); ++, strong (gray scale value: 50–99); or +++, very strong (gray scale value: ≤49). The immunoreactivity scores were obtained by averaging the measurements from each animal.

Results

Delayed neuronal death

To examine ischemia-induced delayed neuronal death in the hippocampus, CV staining was performed. In the sham-operated group, CV-positive cells were observed in all subregions of the hippocampus (Fig. 1A). In the ischemia-operated groups, the distribution pattern of CV-positive cells was not altered at 2 days following ischemia-reperfusion compared with the sham-operated group (Fig. 1B). However, at 4 days following ischemia-reperfusion, a markedly decreased number of neurons in the stratum pyramidale of the CA1 region were stained with CV compared with the sham-operated group (Fig. 1C). This effect was evident only in the stratum pyramidale, as pyramidal neurons in other subregions of the hippocampus were well-stained with CV in the ischemia-operated and the sham-operated groups (Fig. 1C).

Figure 1. CV staining in the hippocampus following transient cerebral ischemia. Hippocampal tissue sections of (A) sham and ischemia-operated groups at (B) 2 days or (C) 4 days post-ischemia were stained with CV (purple signal). A significant loss of CV-positive cells is observed in the stratum pyramidale (marked by asterisk) of the CA1 region at 4 days post-ischemia. These are representative images from the analysis of 7 animals per group. Scale bar, 800 µm (upper panels) and 50 µm (lower panels). CV, cresyl violet; Isch 2 d, day 2 post-ischemia; Isch 4 d, day 4 post-ischemia; CA, conus ammonis; DG, dentate gyrus; SO, stratum oriens; SR, stratum radiatum.

Albumin immunoreactivity

In the CA1 region tissue sections of the sham-operated group, albumin immunoreactivity was observed only in blood vessels, and no albumin-immunoreactive cells were observed (Fig. 2A). However, following transient cerebral ischemia, albumin immunoreactivity was generally increased in the tissue sections and observed in cells. At 2 days after ischemia-reperfusion, albumin immunoreactivity was observed in microglia-like cells in the stratum oriens and stratum radiatum of the CA1 region (Table I and Fig. 2B). At 4 days after ischemia-reperfusion, albumin immunoreactivity was further increased in microglia-like cells in the stratum oriens and stratum radiatum through the CA1 region compared with the sham and the 2 day post-ischemia groups (Table I and Fig. 2C). Notably, at 4 days after ischemia-reperfusion, strong albumin immunoreactivity was also detected in the stratum pyramidale (Table I and Fig. 2C).

Figure 2. Albumin immunohistochemistry in the CA1 region following transient cerebral ischemia. Hippocampal tissue sections of (A) sham and ischemia-operated groups at (B) 2 days or (C) 4 days post-ischemia were immunostained for albumin protein expression (brown signal). Albumin immunoreactivity is observed in hippocampal parenchyma (arrows) as well as perivascular space (arrowheads). Asterisk indicates the albumin immunoreactivity in the stratum pyramidale. These are representative images from the analysis of 7 animals per group. Scale bar, 800 µm (upper panels) and 50 µm (lower panels). Isch 2 d, day 2 post-ischemia; Isch 4 d, day 4 post-ischemia; CA, conus ammonis; DG, dentate gyrus; SO, stratum oriens; SR, stratum radiatum.

Table I. Level of albumin immunoreactivity in microglia of the CA1 hippocampal region following transient cerebral ischemia.

Table I.

Level of albumin immunoreactivity in microglia of the CA1 hippocampal region following transient cerebral ischemia.

	Time after ischemia/reperfusion
Region	Sham	2 days	4 days
SO	−	±	+++
SP	−	−	+++
SR	−	±	+++

[i] Semi-quantification of the immunostaining intensity of albumin was assessed from 6 sections per animal in each group (n=7). Immunoreactivity was scored as follows: negative (−), weakly positive (±), moderate (+), strong (++) and very strong (+++). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

To confirm the identity of the cell type that exhibited specific positive albumin immunostaining in the CA1 region tissue sections at 4 days post-ischemia, double immunofluorescence staining was performed for albumin and Iba-1, an established microglia marker. The results demonstrated that many of the albumin-immunoreactive cells were colocalized with Iba-1-positive microglia (Fig. 3), suggesting that albumin is strongly expressed by microglia in the hippocampal CA1 region following ischemic insult.

Figure 3. Albumin expression in microglia of the CA1 region following transient cerebral ischemia. Hippocampal CA1 region tissue sections from ischemia-operated group at 4 days post-ischemia were stained by immunofluorescence for (A) albumin (red) and (B) Iba-1 (microglia marker, green). (C) Albumin immunoreactivity in Iba-1-positive microglia can be observed in the merged image (arrows). These are representative images from the analysis of 7 animals per group. Scale bar, 100 µm. Iba-1, ionized calcium-binding adapter molecule 1; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Discussion

In the present study, delayed neuronal death in the hippocampal CA1 region following transient cerebral ischemia was examined using CV staining. The results demonstrated that pyramidal neurons in the CA1 region exhibited a ‘delayed neuronal death’ phenotype at 4 days post-ischemic insult. This result is in agreement with the results of previously published studies (1,2,21).

Maeda et al (24) demonstrated that expression of albumin is observed in the extracellular space of the brain parenchyma shortly after transient cerebral ischemia and its prompt accumulation occurred in the periphery of dendrites with neurons, likely due to the process of degeneration and death. In addition, it has been reported that BBB breakdown and extravasation of intravenously injected Evans Blue dye is observed in the forebrain 2 days after ischemic insult, and the extravasation was most severe at 4–6 days following 5 min of transient cerebral ischemia in gerbils (6). Based on these reports, the present study examined albumin immunoreactivity in the hippocampal CA1 region at 2 and 4 days after transient cerebral ischemia. The results demonstrated that albumin immunoreactivity was first observed in Iba-1-immunoreactive microglia near the microvessels in the CA1 region at 2 days after ischemia-reperfusion. In addition, albumin immunoreactivity was further increased in Iba-1-immunoreactive microglia at 4 days after ischemia-reperfusion, and at this time, strong albumin immunoreactivity was also observed in the stratum pyramidale. The present finding is in accordance with previous studies demonstrating that microglia aggregate in the stratum pyramidale of the CA1 region at 4 days after transient cerebral ischemia, the subregion where most of the pyramidal neuron death occurs (23,25).

Albumin is known to induce a unique signaling cascade in microglia, which triggers microglial proliferation (26). Alonso et al (27) reported that extravasated albumin was taken up by activated microglia following ultrasound-mediated BBB opening. However, it has been reported that albumin is synthesized by microglia in the brain as well (28), and that its synthesis and extracellular secretion enhances microglial activation following amyloid β protein fragment 1–42 (Aβ1-42) or lipopolysaccharide-treatment (28). In addition, Byun et al (29) reported that albumin, synthesized mainly from activated microglia, is conjugated with advanced glycation end-product (AGE) to produce a toxic AGE-albumin, which promotes Aβ polymerization and neuronal death. In the present study, ischemia-induced activated microglia were demonstrated to increase albumin immunoreactivity in the CA1 hippocampal region. The underlying mechanism for the increased albumin immunoreactivity observed in microglia only in the CA1 region following transient cerebral ischemia remains unknown. However, based on the above-mentioned literature and the our present study, it can be postulated that albumin in microglia may actively participate in the process of ‘delayed neuronal death’ of the CA1 pyramidal neurons following transient cerebral ischemia. By contrast, albumin is not observed in microglia in striatal parenchyma following thromboembolic middle cerebral artery occlusion in rats (30). Therefore, the expression and function of albumin in microglia may be different in various animal models of ischemia insults and should be investigated further.

In summary, the present study demonstrated that albumin immunoreactivity was newly observed in microglia in the hippocampal CA1 region following 5 min of transient cerebral ischemia. These results indicate that transient ischemia-induced albumin expression in microglia might be associated with ischemia-induced ‘delayed neuronal death’ in the hippocampal CA1 pyramidal neurons.

Acknowledgements

The present research was supported by the Bio-Synergy Research Project (grant no. NRF-2015M3A9C4076322) of the Ministry of Science, ICT and Future Planning through the National Research Foundation, and by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (grant no. NRF-2014R1A1A2058440).

References