Environmental enrichment promotes neural remodeling in newborn rats with hypoxic-ischemic brain damage

Chuanjun Liu, Yankui Guo, Yalu Li, Zhenying Yang

1Department of Pediatrics, Taian Health Centre for Women and Children, Taian 271000, Shandong Province, China

2Central Laboratory of Shandong Agricultural University, Taian 271018, Shandong Province, China

3Morphological Laboratory of Taishan Medical College, Taian 271000, Shandong Province, China

INTRODUCTION

The cerebral cortex is considered equivalent to the hippocampus in terms of functional importance and the anatomical reliance on blood supply[1-3]. The frontal cortex occupies approximately 29% of the total area of the cerebral cortex, and plays an important role in cognitive function[4-5]. As such, we hypothesized that neonatal hypoxic-ischemic brain damage (HIBD) may damage the frontal cortex, resulting in motor and cognitive dysfunctions. We examined neuronal and synaptic structural changes, synaptic numerical density (Nv), and surface density(Sv) in the frontal cortex of Wistar rats after HIBD at postnatal day 7. As early intervention of environmental enrichment (EE) has been shown to improve synaptic deficits in the frontal cortex, we also examined the action of EE intelligence training on neural remodeling in HIBD neonatal rats.

RESULTS

Quantitative analysis of experimental animals

A total of 35 pregnant Wistar rats were included. Rats at postnatal day 7 were randomly divided into three groups with 40 rats in each group. HIBD was established in the model group and EE group, while the sham-operated group received only isolation of the blood vessels. EE intelligence training was then administered from 24 hours to 28 days after modeling, twice per day. Model group and sham-operated group rats were housed in cages with normal feeding. A total of 120 offspring rats were involved in the final analysis.

EE micropathological changes in the frontal cortex in HIBD offspring rats

In brain sections stained with silver nitrate,the sham-operated group exhibited frontal cortex cells that were clear, with irregularly shaped neurons with large nuclei and prominent nucleoli at all recovery periods.Numbers of neurons were increased, nuclei and nucleoli became large with increasing age, and there was an increase in cellular processes, with nerve fiber arranged overlapping, after 14 days recovery (Figures 1A,D).

The model group and the EE group showed similar microscopic findings at 24 hours and 7 days after operation. There was evidence of numerous cells with pyknotic nuclei,empty nests remaining after some cells died,enlargement of the extracellular space and surrounding capillary space, a large number of vacuoles in the cytoplasm, and glial cell nuclei proliferation (Figures 1B-C). As brain edema gradually faded after 14 days of recovery in the model group, the nerve fiber density of the model group increased, but remained discontinuous. Focal proliferation of glial cells was also seen (Figure 1E).

However, the EE group exhibited cells with a clear structure under light microscopy, with a significant improvement in cellular morphology and fiber density compared with those in the model group. There was no difference in neural morphology in the EE group compared with the sham-operated group after 14 days. Nerve tissue and neurons showed a clear structure, nerve fiber morphology was normal, and nerve fiber density was increased (Figure 1F, supplementary Figure 1 online).

EE ultrastructure change of the frontal cortex in HIBD offspring rats

Transmission electron microscopy showed that neurons in sham-operated rats exhibited a clear contour with a large round nuclei and prominent nucleoli, abundant round or oval-shape mitochondria in the cytoplasm,smooth membrane, a symmetrical crest, high levels of rough endoplasmic reticulum, and free ribosomes.

(Figure 2A). The intracytoplasm mitochondria, rough endoplasmic reticulum, and ribosome density increased with age prior to 14 days of recovery, and then gradually stabilized. Cellular processes connected to form neuropils, which contained a large number of synapses.

Several synapses were seen in one visual field, the synaptic structure was intact, and membrane systems(mainly presynaptic membrane and synaptic vesicle membrane) were clear. The gray I postsynaptic membrane compact layer was thick, and electron density was high (dense synaptic vesicles were seen in terminal button) (Figure 2B).

At 24 hours to 7 days after modeling, serious damage to brain tissue was observed in the model group and the EE group. Neurons, glial cells, and capillary gaps were enlarged, and these cells contained low-electron-density flocculent material. Some neurons appeared as vacuoles in the cytoplasm, mitochondria were swollen and even condensed with a fuzzy ridge, rough endoplasmic reticulum was swollen and degranulated, the cisterane was expanded, and nuclei were condensed with an unclear boundary (Figure 2C). The number of synapses in the neuropil was reduced, and the remaining synapses exhibited marked damage; the terminal buttons were unclear, membrane exhibited an uneven thickness or was even dissolved, some areas were not continuous,vacuolated mitochondria were visible, and synaptic vesicles exhibited a vague boundary and even cavity formation (Figure 2D).

At 14 days after modeling, brain edema was significantly attenuated in the model group, with evidence of both neuronal apoptosis and repair. The recovering neurons still exhibited damaged mitochondria and endoplasmic reticulum, gathering of the nuclear chromatin, a high electron density of dense structure in the neuropil, but an increased number of synapses. There were a few vesicles in the terminal button, with an unclear boundary(Figures 2E-F). The morphology of neurons, synaptic density, and structure in the EE group were similar to that in the sham-operated group (Figures 2G-H, supplementary Figure 2 online).

An unilateral grid test system was used to measure the Nv and Sv for each group. Compared with the sham-operated group, the Nv and Sv were significantly reduced in the model group at different time points (P ＜0.05 or P ＜ 0.01). Compared with the model group, the Nv and Sv were increased significantly in the EE group at each time point except at 24 hours (P ＜ 0.05 or P ＜ 0.01;Tables 1-2).

Figure 1 Structure of the frontal cortex in the rat brain (silver nitrate staining, light microscopy, × 400).

Figure 2 Ultrastructure of frontal cortex neurons and synapses (transmission electron microscopy).

Table 1 Changes of synaptic numerical density in the frontal cortex of the rats at different time points after modeling (/μm3)

Table 2 Changes of synaptic surface density in the frontal cortex of rats at different time points after modeling (/μm)

DISCUSSION

It is well established that molecular pathological changes can occur in the brain within 2 hours of HIBD, followed by a rapid increase in morphological changes that peak at 7 days, and then a more delayed phase of cellular apoptosis and repair processes[6-12]. We found that the frontal cortex of rats was markedly damaged in EE and model groups at 24 hours and 7 days after HIBD. Due to loss of projection neurons, the number of normal neurons in the frontal cortex was reduced, and cells and organelles exhibited obvious pathological changes indicative of severe cellular damage[13-14].

The synapse is the key component of brain structure and function, and neural circuits and synapses are in a dynamic state. We found that the total number of synapses was gradually increased during the process of brain development. Synaptic remodeling underlies the neurobiological base for the growth and development of the nervous system, neuronal damage and repair, and learning and memory, and is also involved in many pathophysiological processes such as alcoholism and drug addiction and dependence[15-17]. Synaptic remodeling includes remodeling of function and morphology,which consists of the formation of new synapses, synaptic shape and size, and synaptic density[18-19]. Nv is an indicator of the number of synapses in a certain region,and is used to illustrate changes in synaptic plasticity[20].

Sv is a measure of the area density of the synaptic active zone, and illustrates the total area of the synaptic contact zone in a spatial reference system; a larger area suggests a stronger synaptic function[20]. We found that slightly damaged neurons gradually recovered with increasing time after acute ischemia, and the Nv and Sv increased, although remained lower than the sham-operated group. These findings suggest that both developing and mature brains can exert a strong plasticity and self-repair capacity within a short time after ischemia and hypoxia.

Pu et al[1]reported that early intervention could prevent or reduce cerebral ischemia-induced synaptic ultrastructural injury and promote normal development of synapses. We used offspring rats at postnatal day 7 (P7),P13, P25, and P35 that had equivalent age to the human at perinatal, infant, early childhood, and prepubertal ages,respectively[21-22]. We found that EE intelligence training was able to repair neural tissue injury at different stages of development, with improvement in Nv and Sv to levels similar to the sham-surgery group at 14 days of recovery.

Thus, the improvement in the shape of nerve cells, the increase in numbers of nerve cells, and the increase in nerve fiber density, synaptic number, and synaptic area suggest that EE intelligence training caused synaptic remodeling. These changes may be related to secretion of neurotrophic factors due to enhanced function of astrocytes, improved adaptation of the local microenvironment in the brain, the activation of silent synapses,neural stem cell migration and differentiation, and the formation of new synapses[9,23-26].

In summary, we found that early intervention with EE intelligent training can promote synaptic remodeling in neonatal rats with HIBD.

MATERIALS AND METHODS

Design

A randomized controlled animal experiment.

Time and setting

Experiments were performed from October 2008 to December 2009 at the Central Laboratory in Shandong Agricultural University and the Morphological Laboratory of Taishan Medical College, China.

Materials

A total of 35 healthy Wistar pregnant rats (SPF grade;weighing 300 ± 70 g) were provided by the Experimental Animal Center of Shandong University of Traditional Chinese Medicine, China (License No. of SCXK (Lu)2003-0004). Animals were housed in clean environment,and both male and female rats at postnatal day 7 were included in this study (weighing 13-17 g). All experimental protocols were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals,issued by the Ministry of Science and Technology of the People’s Republic of China[27].

Methods

Modeling and intervention

A median incision was made in the cervical part of rats under ether anesthesia, and the right common carotid artery was separated and ligated. To establish the model,animals were then placed in a closed container with 8%O2and 92% N2for 2 hours after they were incubated for 2 hours[28]. The rats were returned to their mothers after experimentation. At 24 hours after modeling, EE intelligent training was administered to rats, twice per day, as reported[29-31]. To reinforce the stimulus, the training method was modified as follows: the training box(self-made) was encircled by organic glass (70 cm × 60 cm × 60 cm). Objects of different colors, shapes, smells,and textures were placed into the box, such as soft or hard balls, paper boxes, a swing, a seesaw, wood shavings, and essential balm cotton balls. The training was performed under the stimulation of dark or bright red and green lights, as well as gentle music. The training time was 9:00-10:00 a.m. and 4:00-5:00 p.m. The object location and colors were changed weekly. The model group and the sham-operated group were conventionally fed in the cage, with a thin layer of sawdust on the bottom of the cages.

Specimen collection

At 24 hours (postnatal 8 days), 7 days (postnatal 14 days), 14 days (postnatal 21 days), 21 days (postnatal 28 days), and 28 days (postnatal 35 days) after modeling,eight mice from each group underwent a thoracotomy under ether anesthesia, followed by left ventricular puncture. Samples were first rinsed with heparin-contained saline until clear effluent exited the right auricle, followed by 4% paraformaldehyde fixation. Following decapitation, the brain tissues were isolated. The right frontal cerebral cortex near the sagittal suture (3 mm × 2 mm × 2 mm) was cut into about 1 mm3pieces and used for further experiment.

Pathological changes of rat frontal cortex as detected by light microscopy

The perfused frontal cortex samples were immersion fixed with 4% paraformaldehyde for 8-12 hours, and then processed into paraffin sections (10 μm thick). Samples were stained with Bielschowskyis silver nitrate (AgNO3)as follows: 3% AgNO3plating, 10% formaldehyde reduction, then 0.2% gold chloride coloration, dehydration, and transparency, and mounting with neutral gum. Samples were observed under optical microscope (Nikon, Tokyo,Japan).

Ultrastructure of rat brain frontal cortex as detected by transmission electron microscopy

Specimens were fixed with 4% glutaraldehyde and 1%osmium tetroxide. Three embedded ultra-thin slices were randomly selected from each rat at 70 nm thick and made into conventional sections for electron microscopy.

Two copper meshes were selected from each embedded block for observation using a JEM-1200EX transmission electron microscope (Japan Electronics, Tokyo, Japan).

Neuropil areas at 5 000 × magnification were photographed in five visual fields for quantitative analysis.

Morphometric methods

The unilateral grid test system (distance between points = 4 mm; Imaging Institute, Chinese Academy of Military Medical Sciences, Beijing, China) was randomly applied to the photographs to measure the number of drop-off points of the membrane structure (excluding glial cells and capillaries) and the number of synapses in neuropil areas within each test zone. Each photograph was examined in two domains. The synaptic Nv and Sv were calculated according to the formula[20,32]:

where Nxi is the number of synaptic sections, a is the area of each test grid, Z is the length of the test line, Ixi is the number of the cross points between the synapse and the test line, and Pri is the number of reference drop-off points.

Statistical analysis

Data were analyzed with SPSS 11.0 statistical software(SPSS, Chicago, IL, USA) and expressed as mean ± SD.Differences between the groups were compared using the F test, and the SNK method was utilized for multiple comparisons between groups. A level of P ＜ 0.05 was considered statistically significant.

Author contributions:Chuanjun Liu was the project leader,wrote the manuscript, and was responsible for the project design, operative modeling, specimen collection, and section observations. Yankui Guo participated in the electron microscopy sample preparation, observation, and photographing. Yalu Li was responsible for the intelligence training and observation of animals, preparation of histological sections, and image processing. Zhenying Yang assisted the operative modeling and specimen collection, data collation, and analysis.

Ethical approval:This study was approved by the Animal Ethics Committee of Taishan Medical College, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 20, 2011 after selecting the “NRR Current Issue” button on the page.

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