Cell Death/Proliferation and Alterations in Glial Morphology Contribute to Changes in Diffusivity in the Rat Hippocampus after Hypoxia

Abstract

To understand the structural alterations that underlie early and late changes in hippocampal diffusivity after hypoxia/ischemia (H/I), the changes in apparent diffusion coefficient of water (ADC_W) were studied in 8-week-old rats after H/I using diffusion-weighted magnetic resonance imaging (DW-MRI). In the hippocampal CA1 region, ADC_W analyses were performed during 6 months of reperfusion and compared with alterations in cell number/cell-type composition, glial morphology, and extracellular space (ECS) diffusion parameters obtained by the real-time iontophoretic method. In the early phases of reperfusion (1 to 3 days) neuronal cell death, glial proliferation, and developing gliosis were accompanied by an ADC_W decrease and tortuosity increase. Interestingly, ECS volume fraction was decreased only first day after H/I. In the late phases of reperfusion (starting 1 month after H/I), when the CA1 region consisted mainly of microglia, astrocytes, and NG2-glia with markedly altered morphology, ADC_W, ECS volume fraction and tortuosity were increased. Three-dimensional confocal morphometry revealed enlarged astrocytes and shrunken NG2-glia, and in both the contribution of cell soma/processes to total cell volume was markedly increased/decreased. In summary, the ADC_W increase in the CA1 region underlain by altered cellular composition and glial morphology suggests that considerable changes in extracellular signal transmission might occur in the late phases of reperfusion after H/I.

Keywords

astrocytes extracellular volume microglia neurons NG2-glia tortuosity water diffusion

Introduction

Cerebral hypoxia/ischemia (H/I) leads to a sequence of pathophysiological events, such as cell swelling, gliosis, inflammation, and acute and delayed cell death, not only in neurons, but also in adjacent glial cells (Schmidt-Kastner and Freund, 1991; Schmidt-Kastner et al, 1990). Glial cells, namely astrocytes, exhibit characteristic responses to CNS pathology, such as rapid cell swelling occurring immediately after ischemia or trauma, whereas subsequently, reactive gliosis develops within 3 days after insult, characterized by astrocyte proliferation and cellular hypertrophy (Alonso, 2005; Frisen et al, 1995). The participation of NG2-glia in the early events leading to astrogliosis was demonstrated after a cortical stab wound (Alonso, 2005), and their increased proliferation was observed after spreading depression (Tamura et al, 2004). Oligodendrocytes undergo degenerative changes, including the disintegration of myelin, and cell death, whereas microglia become activated, proliferate and change into motile, phagocytic and cytotoxic cells that occupy the site of the injury (Goddard et al, 2002). Such extensive changes in cell number (Pforte et al, 2005), cellular-type composition, cell morphology (Sullivan et al, 2010), and extracellular matrix composition lead to extracellular space (ECS) volume changes (Sykova et al, 2005; van der Toorn et al, 1995) that significantly influence brain diffusivity during the early and late phases of reperfusion.

Diffusion-weighted magnetic resonance imaging (DW-MRI) is an advanced imaging technique that allows the noninvasive evaluation of water diffusivity in brain tissue. Biophysical characteristics, such as the ratio between intracellular and extracellular volumes, the water permeability of cell membranes, the direction of axonal pathways and tissue microstructure, were shown to influence the values of the apparent diffusion coefficient of water (ADC_W) (Gass et al, 2001). In animal stroke models, DW-MRI has been routinely used to demonstrate a decrease in ADC_W during H/I, which is likely related to cytotoxic edema (Dijkhuizen et al, 1998; Vorisek et al, 2002; Vorisek and Sykova, 1997), energy depletion and ionic imbalance (Busza et al, 1992). After the acute phase of ischemia, changes in cellular morphology were shown to underlie ADC_W renormalization in the rat cortex (Zoremba et al, 2008) or dorsal hippocampus and a secondary decrease in ADC_W in the early phases of reperfusion (Li et al, 2002; Lythgoe et al, 2005). However, data showing the time-dependent changes in ADC_W during the early (within days) and late phases (after months) of ischemia/reperfusion are limited, and no attempt has been made to correlate these changes with ongoing cell death/proliferation or with changes in cell-type composition and cell morphology in the region affected by hypoxic—ischemic injury.

Therefore, we have studied the time-dependent changes of ADC_W in the rat hippocampal CA1 region and correlated them with changes in total cell number and cell-type composition in the acute as well as the late stages of reperfusion using a model of cerebral H/I and using DW-MRI and quantitative immunohistochemistry. Extracellular space diffusion parameter measurements using the real-time iontophoretic method were used to clarify the ADC_W changes during reperfusion. Furthermore, three-dimensional confocal morphometry of fluorescently labeled cells was used to reveal changes in the morphology of NG2-glia and astrocytes during reperfusion.

Materials and methods

Induction of Hypoxia/Ischemia

All procedures involving the use of animals were approved by the local ethical review committee and were in agreement with European Communities Council Directive (86/609/EEC). Wistar rats (male, 7 to 9 weeks old; 220 to 260 g) were premedicated with atropin (100 μg/kg, subcutaneously, Biotika, Slovak Republic) and anesthetized with sodium pentobarbital (65 mg/kg, intraperitoneally, Sigma-Aldrich, Prague, Czech Republic). The rats were intubated using a cannula tube (Abbocath-T 16G, Abbott, Sligo, Ireland) and held on mechanical ventilation (33.3% O₂ and 66.6% N₂; rate 60 cycles/min; Animal ventilator CIV-101, Columbus Instruments, Columbus, OH, USA) 15 minutes before carotid artery occlusion and during the first 60 minutes of reperfusion. Hypoxia/ischemia was induced by a bilateral 15 minute occlusion of the common carotides using aneurism clips combined with hypoxic conditions (6% O₂ and 94% N₂, Linde Gas, Prague), as described previously (Dijkhuizen et al, 1998; Knollema et al, 1995). The core temperature was maintained at 37°C with a heating pad throughout the procedure and recovery period until the animals awoke. After the occlusion, the rats were left to survive for 6 hours (6H) 1, 3, 7 days (1D, 3D, 7D), 1, 3, or 6 months (1M, 3M, 6M) before decapitation. Sham-operated animals receiving the same surgical procedure without artery occlusion were used as controls.

Immunohistochemistry

At different time points after H/I, the rats were anesthetized (100 mg/kg sodium pentobarbital, intraperitoneally) and perfused transcardially with 100 mL of saline with heparin (2,500 IU/100 mL; Zentiva, Prague, Czech Republic) followed by 200 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). Brains were dissected out and postfixed in paraformaldehyde solution for 3 hours, then placed in 0.1 mol/L phosphate buffer with gradually increasing sucrose concentrations (10%, 20%, 30%) for cryoprotection. Coronal slices, 30 μm thick, were prepared using a microtome (HM 400, Microm Int. GmbH, Waldorf, Germany). Free-floating sections were incubated with specific antibodies for glial cells and neurons in combination with antibodies specific for apoptosis and proliferation. All immunohistochemical stainings were performed at 4°C in a blocking solution containing 5% Chemiblocker (Millipore, Prague, Czech Republic) and 0.5% Triton X-100 (Sigma-Aldrich) in 0.01 mol/L phosphate-buffered saline. The staining procedure was as follows: the slices were first incubated in the blocking solution for 2 hours, followed by overnight incubation with the primary antibody at 4°C, and subsequent incubation with the appropriate secondary antibody for 2 hours at room temperature. To visualize the cell nuclei, the slices were mounted using Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA, USA). The tissue slices were then examined using an LSM 5 DUO spectral confocal microscope (Zeiss, Prague, Czech Republic). All primary and secondary antibodies, their dilutions and manufacturers are listed in Table 1. To visualize degenerating cells, a polyanionic fluorescein derivate Fluoro-Jade B or ApoTag Red in situ apoptosis detection kit (Chemicon, Temecula, CA, USA) was used.

Table 1

Primary and secondary antibodies used for immunohistochemistry

Antigen	Dilution	Type	Manufacturer	Secondary antibody
Glial cells
GFAP-Cy3	1:800	Mouse IgG	Sigma-Aldrich	NA
S100β	1:200	Mouse IgG	Sigma-Aldrich	GAM 488/594
NG2	1:400	Rabbit IgG	Millipore	GAR 488/594
CD11b	1:200	Mouse IgG	Millipore	GAM 594
APC	1:100	Mouse IgG	Calbiochem	GAM 488

Neurons
NeuN	1:100	Mouse IgG	Millipore	GAM 488/594

Proliferation
PCNA	1:1,000	Mouse IgG	Sigma-Aldrich	GAM 594
Nestin	1:1,000	Mouse IgG	Millipore	GAM 488/594

Apoptosis
Cleaved Casp-3	1:50	Rabbit IgG	Cell Signalling	GAR 488/594
Cleaved PARP-1	1:200	Mouse IgG	Cell Signalling	GAM 488/594

Primary antibodies directed against: APC, adenomatous polyposis coli; Casp-3, the cleaved form of caspase-3; CD11b, integrin Mac-1; GFAP, glial fibrillary acidic protein; NA, not applied; NeuN, neuron-specific nuclear protein; NG2, NG2 chondroitin sulfate proteoglycan; PARP-1, the cleaved form of poly-ADP-ribose polymerase; PCNA, proliferating cell nuclear antigen; S100β, β-subunit of calcium-binding protein.

Secondary antibodies: GAR 488/594: goat anti-rabbit IgG conjugated with Alexa Fluor 488 or 594; GAM488/594: goat anti-mouse IgG conjugated with Alexa Fluor 488 or 594 (all from Molecular Probes). Anti-GFAP is conjugated with Cy3.

Diffusion-Weighted Magnetic Resonance Imaging

The DW-MRI measurements were performed using an experimental magnetic resonance spectrometer Biospec 4.7 T system (Bruker, Ettlingen, Germany) equipped with a 200-mT/m gradient system and a head surface coil. For DW measurements, four coronal slices were selected (thickness, 1.0 mm; interslice distance, 0.5 mm; field of view, 1.92 × 1.92 cm²; matrix size, 256 × 128). The DW images were obtained by using the stimulated echo sequence and the following parameters: b-factors, 136, 329, 675, 1,035, 1,481, and 1,825 s/mm²; Δ=30 milliseconds; δ=5 milliseconds; echo time=46 milliseconds; repetition time=1.2 seconds. The diffusion gradient pointed along the rostrocaudal direction. Maps of ADC_W were calculated by using the linear least-squares method. The ADC_W was assumed to be zero in pixels where the acquired data did not fit well to theoretical dependence (correlation coefficient <0.2). These zero values were ignored for statistical evaluation if they occurred in the region of interest. The ADC_W maps were analyzed using IMAGEJ software (W Rasband, National Institutes of Health, Bethesda, MD, USA). The regions of interest were positioned in both hemispheres according to a rat brain atlas. In each animal, we analyzed one coronal slice (both hemispheres) positioned 3.6 mm caudal to bregma. The resulting values from corresponding regions in the left and right hemispheres were averaged to obtain a single representative value for comparison to other animals. The quality of ADC_W measurements was verified by means of five diffusion phantoms placed on the top of the rat's head. The phantoms were made from glass tubes (inner diameter, 2.3 mm; KS80 glass, Rückl Glass, Otvovice, Czech Republic) filled with pure (99%) substances having different diffusion coefficients. We used the following substances: 1-octanol, n-undecane (Sigma-Aldrich), isopropyl alcohol, 1-butanol, and tert-butanol (Penta, Prague, Czech Republic). The temperature of the phantoms was maintained at a constant 37°C. For DW-MRI measurements, the animals were anesthetized with isoflurane administered by a face mask. The rats were placed in a heated cradle, and their heads were fitted in a built-in head holder (Rapid Biomedical, Rimpar, Germany). The ADC_W was evaluated bilaterally in the rat cortex and hippocampus.

Measurements of Extracellular Space Diffusion Parameters

Measurements of extracellular volume fraction (α; α=volume of the ECS/total volume of the tissue), tortuosity (λ; λ²=D/ADC, where D is the free diffusion coefficient and ADC is the apparent diffusion coefficient) and nonspecific uptake (k′) were performed by the real-time iontophoretic method (Sykova and Nicholson, 2008). In brief, the concentration of tetramethylammonium (TMA⁺), which is administered into the tissue by iontophoresis, was determined using double-barreled ion-selective microelectrodes (TMA⁺-ISMs), filled with an ion exchanger Corning 477317 and as a backfilling solution 100 mmol/L TMA⁺ (Supplementary Figures 1A and 1B). For measurements in a potentially anisotropic medium, the parameters α_x, λ_x, α_y, λ_y, α_z, λ_z were extracted from modified diffusion equations valid for three orthogonal axes x, y, and z. The real value of the scalar variable α was calculated using averaged experimental data from each axis (Sykova and Nicholson, 2008).

The ECS diffusion parameters were determined in vitro in the center of 400 μm thick brain slices. Before slicing, the rats were anesthetized (100 mg/kg sodium pentobarbital, intraperitoneally) and perfused transcardially with 40 mL of ice-cold isolation solution containing (in mmol/L): 110 NMDG-Cl, 3 KCl, 23 NaHCO₃, 1.25 Na₂HPO₄, 0.5 CaCl₂, 7 MgCl₂, 20 glucose (pH 7.4, 290 mOsm/kg). Brains were dissected out and coronal and sagital slices were prepared using a microtome (HM 400, Microm Int. GmbH). After dissection, slices were maintained for 30 minutes in isolation solution at 34°C and then kept in artificial cerebrospinal fluid. Recordings were performed at room temperature (22°C to 26°C) in a chamber perfused with a continuously bubbled (95% O₂ and 5% CO₂) artificial cerebrospinal fluid containing in mmol/L: 117 NaCl, 3 KCl, 35 NaHCO₃, 1.25 Na₂HPO₄, 1.3 MgCl₂, 1.5 CaCl₂, 10 glucose, and 0.1TMA⁺ (pH 7.4, ∼300 mOsm/kg) at a flow rate of 10 mL/min. In each slice, the measurements were performed in two to three different tracks in the CA1 stratum radiatum; data extracted from three diffusion curves were averaged in each individual electrode track.

Confocal Morphometry

Changes in astrocyte and NG2-glia morphology were determined from three-dimensional images of cells filled with Alexa-Fluor-hydrazide 488 or 594 as described previously (Benesova et al, 2009; Chvatal et al, 2007) using a LEICA TCS SP confocal microscope equipped with an Arg/HeNe laser. Briefly, the cells were recorded as a set of ∼250 to 300 consecutive two-dimensional images with a uniform spacing of 0.12 μm. The resolution of two-dimensional images was 1,024 × 1,024 pixels. The cell surface was found in each image using an edge-detecting algorithm, and the area of the image surrounded by the edge was calculated for each layer. The values of cell volume for individual cells were obtained by integrating the values of the edge length and area from all images in a set (Supplementary Figures 1C–1E). Image processing and morphometric measurements were performed using the program CellAnalyst developed in the Institute of Experimental Medicine, Prague, Czech Republic. The following parameters were compared: total cell volume (V), volume of the cell soma (V _s), volume of the cell processes (V _p), and the ratio of either V _s or V _p to V (V _s/V, V _p/V).

Changes in Cell Number After Hypoxia/Ischemia Followed by Reperfusion

Changes in cell number due to cell death/proliferation were quantified by analyzing the number of cells in the hippocampal CA1 region in rats after H/I and comparing that to the number of cells in the same region in sham-operated animals. Brain slices from sham-operated animals were analyzed at three different time points—7D, 1M, and 6M after H/I. We used two rats for each time point of reperfusion, and four consecutive coronal sections (40 μm thick) of a hippocampal segment (from bregma- caudally 3.3 to 3.6 mm) were analyzed from each rat. From each coronal brain section, eight images (225 × 225 × 20 μm³, step size 1 μm) of the hippocampal CA1 region covering the stratum radiatum, pyramidal cell layer, and stratum oriens were taken from both hemispheres using a LSM 5 DUO spectral confocal microscope (Zeiss). The regions selected for quantifying changes in cell number are shown in the insets of Figure 4A. The number of DAPI-, neuron-specific nuclear protein (NeuN)-, proliferating cell nuclear antigen (PCNA)-, adenomatous polyposis coli (APC)-, integrin Mac-1 (CD11b)-, β-subunit of calcium-binding protein (S100β)-, glial fibrillary acidic protein (GFAP)-, and NG2-positive cells was estimated from superimposed images using GSA Image Analyzer v3.0.5. (Digital River, Eden Prairie, MN, USA). The changes in cell number were expressed either as the number of cells per section of 225 × 225 × 20 μm³ or as the percentage of NG2/PCNA- and S100β/PCNA-positive cells from the total number of NG2- and S100β-positive cells. The percentage of neurons and glial cells in the hippocampal region of control rats and those after H/I was expressed as the number of NeuN-, S100β-, APC-, CD11b-, and NG2-positive cells out of the total number of DAPI-positive cells.

Figure 4

Time-dependent changes in the cellular composition of the hippocampal CA1 region after hypoxia/ischemia (H/I) due to increased cell death/proliferation. (A) Time-dependent changes in the number of DAPI (4′,6-diamidino-2-phenylindole) and proliferating cell nuclear antigen (PCNA)-positive cells per section (225 × 225 × 20 μm³). (B) Time-dependent changes in neuron-specific nuclear protein (NeuN)- and CD11b-positive cells. (C) Changes in the number of NG2-, glial fibrillary acidic protein (GFAP)-, and adenomatous polyposis coli (APC)-positive cells. (D) Time-dependent changes in the number of PCNA⁺ astrocytes and PCNA⁺ NG2-glia expressed as a percentage of the total number of astrocytes or NG2-glia counted in the CA1 region. Note that almost 40% of NG2-glia and only 6% of astrocytes in the CA1 region proliferated 3 days after H/I. Selected regions, where the cell counting was performed, are shown in the inset. Two rats were used for each time point, two slices were taken for analysis from each animal (from bregma-caudally 3.3 to 3.6 mm) and 8 selected regions (225 × 225 × 20 μm³) were analyzed from each slice. All together, for each time point 32 hippocampal regions were analyzed. The following abbreviations were used: CTRL (control), 6H (6 hours), 1D (1 day), 3D (3 days), 7D (7 days), 1M (1 month), 3M (3 months) and 6M (6 months). Cell counts were corrected for hippocampal volume shrinkage 3 and 6 months after H/I.

Statistical Analysis

The results are expressed as the mean±s.e.m. Statistical analysis of the differences between groups was evaluated using one-way analysis of variance for multiple comparisons with Tukey's post hoc analysis. Values of P<0.05 were considered significant, P<0.01 were considered very significant, and P<0.001 were considered extremely significant.

Results

We have combined immunohistochemistry, cell morphometry, and DW-MRI to reveal the main cellular contributors to altered diffusivity in the nervous tissue in response to H/I. Neuronal/glial markers combined with specific markers for apoptosis and proliferation were used to determine the changes in the number of neurons, astrocytes, NG2-glia, oligodendrocytes, and microglia in the rat hippocampus at different time points: 6H, 1D, 3D, 7D, 1M, 3M, and 6M after H/I. The alterations in cell number and cell-type ratio together with the changes in glial cell morphology were correlated with the changes in ADC_W obtained by DW-MRI and the changes in the ECS volume fraction α and tortuosity λ measured by the TMA⁺ method after 1D, 3D, and 1M of reperfusion. For DW-MRI measurements, immunohistochemical analysis and cell morphometry, H/I was induced in 68 rats. Sham-operated rats were used as controls (n=34). Nonoperated rats (n=8) were used for comparing the values of ADC_W with those obtained in sham-operated animals. For TMA⁺ method measurements, 6 sham-operated and 10 H/I rats were used.

Immunohistochemistry of the Rat Hippocampus After Hypoxia/Ischemia

Similarly to previous findings, H/I resulted in marked immunohistochemical changes in the hippocampal CA1 subfield (Dijkhuizen et al, 1998; Kuan et al, 2004; Schmidt-Kastner and Freund, 1991). The NeuN immunoreactivity decreased within the first 3 days after H/I, and no recovery of NeuN staining was observed within the next 3 to 6 months (Figures 1A–1F). As described previously, increased GFAP immunoreactivity was detected in the hippocampal CA1 region, the subiculum, and the polymorph layer of the dentate gyrus (Schmidt-Kastner et al, 1990). A transient increase in GFAP immunoreactivity was also observed in the primary somatosensory cortex—barrel field (data not shown). One month after H/I, increased GFAP immunoreactivity was only detected in the hippocampal CA1 region and the subiculum, and it remained elevated in these regions for another 5 months (Figures 1E and 1F). In the acute phases of reperfusion (6H and 1D after H/I), hippocampal astrocytes displayed thin long processes (Figure 1B), whereas 3 and 7D after H/I, the typical hypertrophied astrocytes with thickened processes and enlarged cell bodies appeared in both hippocampi (Figures 1C and 1D). The reactive astrocytes coexpressed nestin, an early marker of astrocyte activation (Frisen et al, 1995). A striking increase in nestin/GFAP coexpression was observed 3D, 7D, and 1M after H/I, then it started to decline after 3 months of reperfusion (Figures 1A–1F). In addition, nestin-positive NG2-glia were detected in all regions of the CA1 hippocampus starting 1D after H/I (data not shown).

Figure 1

Immunohistochemical analyses of the rat hippocampus after hypoxia/ischemia (H/I) followed by reperfusion. Coronal sections of the rat hippocampus (left) immunostained for neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP) in controls (A) and 1 day (B), 3 days (C), 7 days (D), 1 month (E), and 3 months after H/I (F). Enlargements of the tissue sections shown on the right illustrate changes in immunoreactivity for NeuN, GFAP, and nestin in the hippocampal CA1 region. Yellow color indicates double-stained GFAP/nestin-positive cells. The following abbreviations were used: CTRL (control), 1D (1 day), 3D (3 days), 7D (7 days), 1M (1 month), and 3M (3 months) after H/I.

As shown previously by Beilharz et al (1995), activated, CD11b-positive microglia were detected in the CA1 region and the polymorph layer of the dentate gyrus from 1D after H/I onwards, and within 7 days they occupied the entire CA1 region (Supplementary Figures 2A–2C). Increased CD11b immunoreactivity persisted in the hippocampal CA1 region 1M after H/I, whereas after 3M and 6M it declined. In addition, an antibody against NG2 chondroitin sulfate proteoglycan was used to visualize the changes in NG2-glia morphology in response to H/I. Increased NG2 immunoreactivity was observed in the CA1 region and dentate gyrus 3, 7D and 1M after H/I, then it started to decline (Supplementary Figures 2C–2E).

Time-Dependent Changes in the Apparent Diffusion Coefficient of Water and Extracellular Space Diffusion Parameters After Hypoxia/Ischemia

The DW-MRI measurements of ADC_W were performed bilaterally in the CA1 region of the hippocampus and the dentate gyrus as well as in the primary motor and somatosensory cortex and the corpus callosum, where an increase in GFAP staining was also observed. Typical DW-MRI images are shown in Figure 2A. We found no significant differences between the values of ADC_W obtained in the cortex or hippocampus of sham-operated rats at different time points after H/I (6H, 3D, 1M, 3M, and 6M), and moreover, they were comparable with those measured in nonoperated animals (Supplementary Figures 3A and 3B). Therefore, all sham-operated animals were pooled and used as controls in Figure 2B.

Figure 2

Time-dependent changes in the apparent diffusion coefficient of water (ADC_W) in the rat brain after hypoxia/ischemia (H/I). (A) Typical ADC_W maps of a control rat brain (sham-operated animal) and of a rat brain 1 day and 1 month after H/I. (B) Time-dependent changes in ADC_W in the CA1 region of the hippocampus. The selected region used for ADC_W analyses is highlighted by red color in a T2-weighted image of the rat brain. ADC_W was analyzed bilaterally in both hippocampi. (C) Changes in extracellular volume fraction α and tortuosity λ in the hippocampal CA1 subfield (measured in situ) 1D, 3D, and 1M after H/I. Note that 1D after H/I ADC_W decrease correlates with a decrease in α and an increase in λ, whereas the ADC_W increase 1M after H/I correlates mainly with an α increase. The following abbreviations were used: C (control), 6H (6 hours), 1D (1 day), 3D (3 days), 7D (days), 1M (1 month), 3M (3 months), and 6M (6 months).

In the CA1 region of the hippocampus, a marked decrease in ADC_W was detected 1D and 3D after H/I when compared with controls, which is in agreement with previously published data (Dijkhuizen et al, 1998). Subsequently, ADC_W renormalized 7D after H/I. Interestingly, a significant increase in ADC_W was detected in the hippocampal CA1 region 1M and 3 to 6M after H/I (Figure 2B; Supplementary Table 1). The ADC_W decrease detected 1D after H/I was accompanied by a decrease in α and an increase in λ_x,y,z, whereas 3D after H/I, the ADC_W decrease coincided with renormalized α and increased λ_x,y,z. A significant increase in ADC_W starting 1M after H/I was accompanied by a marked increase in α and increased λ_x,y,z (Figure 2C; Supplementary Table 2). As there were no significant differences between ECS diffusion parameters in sham-operated rats 3D and 1M after H/I, they were pooled and used as a control. We did not find any anisotropy in the CA1 stratum radiatum (tortuosity values along the x, y, and z axes were not significantly different in any studied animal group). Similarly to the CA1 region, in the primary motor and somatosensory cortex, ADC_W decreased 1D after H/I. In the dentate gyrus, 6H and 1D after H/I, the ADC_W significantly decreased. Starting 3 days after H/I, the values of ADC_W in the primary motor and somatosensory cortex and the dentate gyrus were not significantly different from those obtained in control rats. Similarly to the hippocampal CA1 region, an increase in ADC_W was detected in the corpus callosum 3 to 6 M after H/I. All ADC_W values obtained in the rat hippocampus, cortex, and corpus callosum are listed in Supplementary Table 1.

Cell Death/Proliferation Induced by Hypoxia/Ischemia in the Rat Hippocampal CA1 Region

As MRI measurements and the TMA⁺ method revealed marked changes in the hippocampal CA1 region diffusivity, we quantified the changes in cell number caused by cell death and cell proliferation, which might also contribute to these alterations.

As described previously, cell death/proliferation occurred in the posthypoxic/ischemic CA1 region and affected neurons as well as glial cells (Beilharz et al, 1995; Fortuna et al, 1997; Kuan et al, 2004; Sas et al, 2008). Six hours after H/I, cleaved caspase-3 was detected in pyramidal neurons of the CA1 hippocampal region followed by the appearance of cleaved poly-ADP-ribose polymerase-1 and resulting in neuronal death 7 days after H/I as visualized by Fluoro-Jade B staining and the terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling (TUNEL) assay (Figures 3A–3D), an immunohistochemical pattern described previously (Kuan et al, 2004). Despite the fact that astrocytes displayed increased immunoreactivity for cleaved caspase-3 in the early stages of reperfusion, Fluoro-Jade B- or TUNEL-positive astrocytes were not detected within 7 days of reperfusion. However, Fluoro-Jade B- and TUNEL-positive hippocampal astrocytes were found 1M and 3M after H/I in the vicinity of the CA1 pyramidal layer. We did not find any APC/TUNEL-positive cells within the hippocampal CA1 region. On the basis of PCNA immunoreactivity, extensive proliferation occurred 3 to 7 days after H/I, predominantly in the CA1 region, the subiculum, and the dentate gyrus, where at this time point, the majority of PCNA-positive cells were CD11b-positive microglia (Figures 3E and 3F). Besides proliferating microglia, also PCNA-positive NG2-glia and PCNA-positive astrocytes were detected in the polymorph layer of the dentate gyrus, the subiculum, the pyramidal layer, the stratum radiatum, and the stratum oriens during the acute as well as the late stages of reperfusion (Figure 3G).

Figure 3

Apoptosis/proliferation in the hippocampal CA1 region after hypoxia/ischemia (H/I). (A, B) The brain tissue sections illustrate changes in immunoreactivity for cleaved caspase-3 (Casp3, A) and the cleaved form of poly-ADP-ribose polymerase (PARP-1, B) in the hippocampal CA1 region 6 hours and 7 days after H/I. Note that 6 hours after H/I, mainly the pyramidal neurons are Casp3 and PARP-1 positive, whereas after 7 days, astrocytes are positive for both apoptotic markers. (C) A coronal section of the rat hippocampus 7 days after H/I immunostained for apoptotic cells using the terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate-biotin nick end labeling (TUNEL) assay. Higher magnification of TUNEL labeling in the CA1 region and glial fibrillary acidic protein (GFAP) illustrates apoptotic pyramidal neurons 7 days after H/I, whereas TUNEL/GFAP-positive astrocytes that appeared 1 month after H/I are shown in the inset. Enlargements of the tissue shown in (D) demonstrate the time-dependent changes in Fluor-Jade B staining (FJB) after H/I: from the left, damaged neurons after 6 hours and 3 days of reperfusion and FJB-positive astrocytes in the vicinity of the pyramidal layer 1 month after H/I. (E) Coronal section of the rat hippocampus 7 days after H/I immunostained for proliferating cell nuclear antigen (PCNA). (F) Enlargements of the tissue sections illustrate increased immunoreactivity for PCNA (left) and CD11b (right) in the CA1 region of the hippocampus after 7 days of reperfusion. (G) A detailed image of a PCNA-positive astrocyte (left) and PCNA-positive NG2-glia (right). The following abbreviations were used: 6H (6 hours), 3D (3 days), 7D (7 days), and 1M (1 month).

DAPI-, PCNA-, NeuN-, S100β-, NG2-, APC-, and CD11b-positive cells were counted in the CA1 region-stratum pyramidale and the adjacent stratum radiatum and stratum oriens to quantify the changes in cell number after H/I (Figures 4A–4D). As we found no significant differences between the numbers of DAPI-, PCNA-, GFAP-, NG2-, CD11b-, and APC-positive cells in the hippocampus of sham-operated rats at different time points after H/I (1D, 1M, 6M), all sham-operated animals were pooled and used as controls. Counting DAPI-positive cells revealed a marked increase in total cell number in the CA1 region of the hippocampus 3D, 7D, and 1M after H/I, caused mainly by the massive proliferation of microglia. The number of microglia was increased from 1D onwards, reaching a maximal number 3 to 7D after H/I, then started to decline after 1 month of reperfusion, but still remained elevated above control values 6M after H/I (Figure 4B).

On the other hand, the number of NeuN-positive cells decreased within the first 7 days of reperfusion, approximately by ∼75%, and additional decreases after 1 and 3 months resulted in an ∼90% neuronal reduction when compared with control rats (Figure 4B). The number of astrocytes decreased 6H after H/I, whereas 1, 3, and 6M after H/I, it was noticeably increased. The oligodendrocyte number (APC-positive cells) declined 6H and 1D after H/I, whereas after 7 days it first increased and then decreased below the numbers found in controls. A decrease in the number of NG2-glia was observed only 7D after H/I, coinciding with an increased number of APC-positive oligodendrocytes (Figure 4C).

Besides microglia, which significantly contribute to increased cell numbers in the CA1 region, NG2-glia were also found to increasingly proliferate. Approximately ∼20% of the total number of NG2-positive cells were proliferating 1D after H/I, and this proportion raised to ∼40% after 3 days of reperfusion. Later, NG2-glia proliferation started to decline, but still remained increased above the control values (Figure 4D). Increased proliferation of astrocytes was detected 3D, 7D, and 1M after H/I, but only 5% to 7% of the total number of astroglia were PCNA-positive (Figures 4C and 4D). Taken together, within 6 months of reperfusion, neuronal cell death and glial cell proliferation resulted in a marked reorganization of the hippocampal CA1 region, where astrocytes and microglia were the most prevalent cell types. Using MRI, we found that the size of the hippocampal CA1 region declined 3M and 6M after H/I by ∼26% in the dorsoventral direction; cell counts were corrected accordingly (Supplementary Figure 4). In the rostrocaudal direction, we observed no significant shrinkage (data not shown).

Morphological Changes of Hippocampal Glial Cells After Hypoxia/Ischemia

Besides continuing cell proliferation as well as cell death in the hippocampal CA1 region, H/I also initiated considerable changes in glial cell morphology. Therefore, we generated three-dimensional images of Alexa-Fluor-hydrazide-labeled astrocytes and NG2-glia to calculate their total cell volume and also to compare the contribution of the cell soma and cell processes to total cell volume in astrocytes or NG2-glia of control rats and those 3D and 1M after H/I (Figures 5A–5D). Three days after H/I, the average values of total astrocyte volume (V), the volume of the processes (V _p), and the volume of the cell soma (V _s) were not significantly different from those obtained in the CA1 region of control animals; however, there was a marked increase in V, V _s, and V _p after 1 month of reperfusion. In addition, the differences in reactive astrocytes from the acute and late phases of reperfusion became more obvious when their compartments, that is the volume of the processes (V _p) and the volume of the cell soma (V _s), were compared and expressed as a percentage of total cell volume. In controls, an astrocytic cell soma occupied ∼20% of the total cell volume and ∼80% was taken up by the cell processes; however, 1M after H/I, the astrocytic cell soma comprised ∼40% of the total cell volume and 60% consisted of the cell processes (Figures 5A and 5B). Compared with NG2-glia from control rats, the average V and V _p of NG2-glia were reduced 3D and 1M after H/I, whereas V _s remained unchanged. Similarly to astrocytes, the contribution of V _s to V in NG2-glia increased as the cell processes retracted (Figures 5C and 5D). In controls, the cell soma occupied ∼30% of the total cell volume and ∼70% was taken up by cell processes, whereas 1M after H/I, the cell soma of NG2-glia made up ∼55% of the total cell volume and the cell processes 45% (Figures 5C and 5D).

Figure 5

Changes in astrocyte and NG2-glia morphology evoked by hypoxia/ischemia (H/I) in the hippocampal CA1 region. (A) Superimposed confocal images of CA1 hippocampal astrocytes in control rats (top), 3 days (middle) and 1 month after H/I (bottom). (B) Top: The time-dependent changes in total astrocyte volume (V) and the volumes of the astrocyte soma (V _s) and processes (V _p). Bottom: The volumes of the cell soma and processes expressed as a percentage of total cell volume. Asterisks (∗) indicate significant differences between astrocytes from control and ischemic rats, whereas crosshatches (#) indicate significant differences between astrocytes 3 days and 1 month after H/I. (C) Superimposed confocal images of CA1 hippocampal NG2-glia in control rats (top), 3 days (middle), and 1 month after H/I (bottom). (D) Top: The time-dependent changes in total NG2-glia volume (V) and the volumes of the cell soma (V _s) and processes (V _p). Bottom: The volumes of the soma and processes of NG2-glia cells expressed as a percentage of total cell volume. Asterisks (∗) indicate significant differences between NG2-glia from control and ischemic rats, whereas crosshatches (#) indicate significant differences between NG2-glia 3 days and 1 month after H/I.

In summary, 1M after H/I, the reactive astrocytes were significantly enlarged, whereas NG2-glia showed a reduced total cell volume during reperfusion. In both cell types, the percentage contribution of the cell soma markedly increased.

Discussion

The early phases of reperfusion after transient H/I (1 and 3 days after H/I) are characterized by an ADC_W decrease in the CA1 region of the hippocampus, and it coincides with considerable glial cell proliferation, marked neuronal cell death, and developing reactive gliosis. We have demonstrated that 1 day after H/I, the ADC_W decreases in correlation with decreased ECS volume fraction and increased tortuosity, whereas after 3 days of reperfusion, the ADC_W decreases rather due to increased tortuosity. In the late phases of reperfusion (1, 3, and 6 months after H/I) alterations in the cellular components, marked changes in astrocyte and NG2-glia morphology and an increased number of activated microglia in the hippocampal CA1 region are accompanied by a significantly increased ADC_W, correlating with increased extracellular volume fraction and tortuosity 1 month after H/I. Using three-dimensional confocal morphometry, we have shown that the morphology of astrocytes and NG2-glia was significantly altered 1 month after H/I: the astrocytes were enlarged, whereas the size of NG2-glia decreased. In addition, the contribution of the cell soma to the total cell volume significantly increased in both astrocytes as well as NG2-glia.

Hypoxia—ischemia has been previously shown to lead to the selective cell death of CA1 pyramidal neurons as well as neurons in the hillus of the dentate gyrus after 48 to 72 hours of reperfusion and to the appearance of reactive astrocytes and activated microglia at the site of injury within 3 to 7 days (Fortuna et al, 1997; Gehrmann et al, 1992; Kuan et al, 2004). Similarly to other types of CNS injury (Anderova et al, 2004; Frisen et al, 1995), the reactive astrocytes in the CA1 hippocampal region were nestin-positive; nevertheless, we have detected a large number of nestin-positive NG2-glia, and their appearance coincided with their increased proliferation. Increased NG2-glia proliferation was described in a model of spinal cord injury and H/I of the neonatal cortex (Lytle and Wrathall, 2007; Sizonenko et al, 2008; Zhao et al, 2009).

Our previous findings demonstrated that ADC_W mirrors changes in extracellular volume fraction and geometry (Vorisek and Sykova, 2009), thus reflecting cell swelling (Vorisek and Sykova, 1997), changes in cell morphology, and the composition of the extracellular matrix (Sykova et al, 2005; Vorisek et al, 2002). There is also evidence that ADC_W has a significant intracellular component (Silva et al, 2002); therefore, the down/upregulation of different intermediate filaments together with structural reorganization of the cells might significantly contribute to total ADC_W. A decrease in the extracellular volume fraction and a corresponding decrease of ADC_W in the brain during H/I is a well-described phenomenon (Dijkhuizen et al, 1998) as is the rapid renormalization of the ECS size after reperfusion (Dijkhuizen et al, 1998; Zoremba et al, 2008). It accords well with our finding that the ADC_W values obtained 6 hours after H/I in the CA1 region (Figure 2B; Supplementary Table 1) were comparable to those obtained in controls. Only in the dentate gyrus was a significant ADC_W decrease detected, possibly reflecting the greater sensitivity of this region to reperfusion and thus the rapid onset of microglia activation, astrogliosis, and NG2-glia proliferation (unpublished data).

An Increasing Number of Glial Cells in the Hippocampal CA1 Region Contribute to an Apparent Diffusion Coefficient of Water Decrease During the Early Phases of Reperfusion

The reappearance of reduced ADC_W 1 to 3 days after H/I due to cytotoxic edema was shown previously (Dijkhuizen et al, 1998). Their findings accord well with our data demonstrating decreased ADC_W, significantly reduced ECS volume fraction and increased tortuosity in the hippocampal CA1 region 1 day after H/I. Besides cytotoxic edema, at this time point, the total number of cells already increased due to NG2-glia and microglia proliferation and, therefore, it might also contribute to decreased ADC_W and ECS volume fraction. The proliferation of NG2-glia and microglia peaked 3 days after H/I leading to a marked increase (∼37%) in the total cell number in the CA1 region (Supplementary Figure 5) and to increased expression of NG2 chondroitin sulfate proteoglycan (Supplementary Figure 2). Therefore, the observed ADC_W decrease coinciding with increased tortuosity might partially result from elevated total cell numbers and elevated extracellular matrix protein expression as shown after a cortical stab wound (Vorisek et al, 2002). On the basis of three-dimensional confocal morphometry, neither astrocytes nor NG2-glia showed a marked cell volume increase 3 days after H/I; the later cell type even underwent a cell volume decrease. Changes in glial cell morphology, namely in astrocytes, have been observed during hypoosmotic stress, oxygen—glucose deprivation, and ischemia (Benesova et al, 2009; Hirrlinger et al, 2008; Risher et al, 2009), demonstrating astrocytic swelling during the pathological conditions followed by a recovery phase (within minutes); however, data describing the morphological changes of glial cells in the early/late phases of reperfusion (days/months) are limited. Recently, a retraction of astrocytic processes and a reduction of astrocytic size have been demonstrated in response to H/I followed by 3 days of reperfusion (Sullivan et al, 2010). These results accord well with our data showing an enlarged cell soma (forming ∼27% of the total astrocyte volume) and reduced cell processes 3 days after H/I. Also, the microglial morphology at this time point displays marked process reduction/retraction; therefore, the decrease in ADC_W can be partially caused by increasing glial cell numbers rather than by altered glial morphology.

Interestingly, 7 days after H/I, ADC_W in the cortex and hippocampus renormalized and even increased above control values in the CA1 region of the hippocampus. The renormalized ADC_W in the CA1 region possibly results from extensive neuronal cell death: at this time point the number of neurons decreased by ∼75%; however, the ADC_W renormalization might be also caused by a neuronal shrinkage accompanying apoptosis (Ray et al, 2006). We hypothesize that increased ECS volume fraction due to a markedly reduced number of neurons and increased tortuosity due to astrogliosis might lead at this time point to ADC_W renormalization (Vorisek and Sykova, 2009).

Changes in the Cellular Components of the Hippocampal CA1 Region Together with Changes in Glial Cell Morphology Result in an Apparent Diffusion Coefficient of Water Increase During the Late Phases of Reperfusion

Starting 1 month after H/I, the ADC_W in the CA1 hippocampal region was significantly increased when compared with controls. Quantifying the changes in cell numbers with respect to the cell types revealed that the CA1 region mainly consisted of microglia, astrocytes, and NG2-glia 1 month after H/I, whereas the number of neurons was extremely reduced (Supplementary Figure 5). Besides microglia, the number of astrocytes was markedly increased, forming ∼30% of the total cell number. Astrocytes have been shown previously to proliferate in response to injury (Sizonenko et al, 2008); however, compared with the marked microglia and NG2-glia proliferation, the number of proliferating astrocytes was negligible (∼8% of total astrocytes). Despite the fact that a large number of nestin-positive NG2-glia appeared in the CA1 region of the hippocampus in the early stages of reperfusion and that 40% of them were immunoreactive for PCNA, the number of NG2-glia did not significantly increase but rather declined 3 and 7 days after H/I. On the basis of recently published data (Zhao et al, 2009), a subpopulation of reactive astrocytes in the CA1 region might be derived from NG2-glia. Although NG2-glia in normal developing or pathologic brains have been shown to be involved in the genesis of oligodendrocytes (Lytle et al, 2009) or neurons (Yokoyama et al, 2006), the total number of oligodendrocytes increased significantly only 7 days after H/I, and subsequently we observed a decline in their number during reperfusion.

In addition, the size of astrocytes and NG2-glia and their morphology were significantly changed after H/I. We have demonstrated that reactive astrocytes after 1 month of reperfusion were significantly enlarged and that the cell soma comprised about 40% of the total astrocyte volume, whereas in controls, their cell somas formed ∼18% of the total cell volume, a value comparable with that described previously (Chvatal et al, 2007). Although the total astrocytic volume markedly increased, especially the volume of the cell soma, the contribution of the astrocytic processes to the total cell volume significantly decreased. In contrast, the size of NG2-glia diminished; however, they also displayed an enlarged cell soma and reduced/retracted cell processes. Similarly, Sullivan et al (2010) demonstrated in neonatal piglets that cortical astrocytes reduce the number of their primary, secondary, and tertiary processes, diminish the length of their primary processes, and increase the volume of their cell somas in response to H/I. Thus, we hypothesize that starting 1 month after H/I, the cellular composition of the hippocampal CA1 region and glial cell morphology are two important factors contributing to the ADC_W and ECS volume fraction increase detected in this region (Supplementary Figure 5). As astrocytic and NG2-glial processes form a large number of diffusion obstacles in the ECS, any alteration in cell morphology due to cell process retraction and changes in the cell soma/cell processes ratio might be one of the major structural factors contributing to the increased tissue diffusivity observed in the late phases of reperfusion. However, the tortuosity increase was less pronounced than in the acute phase of reperfusion, possibly due to the concomitant ECS volume increase.

In addition to all of the changes occurring in the ECS in terms of, for example, cellular composition, cellular morphology, and the expression of different extracellular matrix proteins, the contribution of the intracellular space of activated glial cells with their altered expression of nestin, GFAP, and vimentin or of apoptotic neurons to total ADC_W might be substantial. Such cytoskeletal remodeling might also significantly contribute to the intracellular component of ADC_W and may have a significant role during H/I and reperfusion.

The correlation of ADC_W/ECS diffusion parameter changes with events such as cell swelling, cell death/proliferation, changes in cellular components, and glial morphology together with changes in extracellular matrix proteins, changes in the intracellular space, and blood—brain barrier disruption, could bring important findings that may improve the quality of diagnosis and outcome prediction in patients after cerebral ischemia.

Footnotes

Acknowledgements

The authors thank Helena Pavlikova and Hana Hronova for immunohistochemical staining.

The authors declare no conflict of interest.

Notes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website ()

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Supplementary Material

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Cell Death/Proliferation and Alterations in Glial Morphology Contribute to Changes in Diffusivity in the Rat Hippocampus after Hypoxia—Ischemia

Abstract

Keywords

Introduction

Materials and methods

Induction of Hypoxia/Ischemia

Immunohistochemistry

Diffusion-Weighted Magnetic Resonance Imaging

Measurements of Extracellular Space Diffusion Parameters

Confocal Morphometry

Changes in Cell Number After Hypoxia/Ischemia Followed by Reperfusion

Statistical Analysis

Results

Immunohistochemistry of the Rat Hippocampus After Hypoxia/Ischemia

Time-Dependent Changes in the Apparent Diffusion Coefficient of Water and Extracellular Space Diffusion Parameters After Hypoxia/Ischemia

Cell Death/Proliferation Induced by Hypoxia/Ischemia in the Rat Hippocampal CA1 Region

Morphological Changes of Hippocampal Glial Cells After Hypoxia/Ischemia

Discussion

An Increasing Number of Glial Cells in the Hippocampal CA1 Region Contribute to an Apparent Diffusion Coefficient of Water Decrease During the Early Phases of Reperfusion

Changes in the Cellular Components of the Hippocampal CA1 Region Together with Changes in Glial Cell Morphology Result in an Apparent Diffusion Coefficient of Water Increase During the Late Phases of Reperfusion

Footnotes

Acknowledgements

Notes

References

Supplementary Material