The effect of Brucella abortus on glial activation and cell death in adult male rat's hippocampus

A zoonotic disease called brucellosis can cause flu-like symptoms and heart inflammation. The bacteria responsible for this disease can also enter the brain, causing a condition called neurobrucellosis that can result in long-term neurological problems. In this study, researchers aimed to determine the changes in the hippocampal cells of rats infected with Brucella . For the study, 24 adult male albino rats were inoculated with 1 × 10 6 CFU Brucella abortus 544. The rats were then deeply anesthetized, and their hippocampus samples were taken for stereological, histological, and molecular studies. The results showed that the infected rats had increased microgliosis and astrogliosis. Furthermore, a high level of caspase-3 in their hippocampal tissue indicated their susceptibility to apoptosis. Additionally, there was a decrease in expression of Ki67, which further supported this. Sholl's analysis confirmed a significant failure in glial morphology. The study demonstrated that the pathogen has the ability to destroy the hippocampus and potentially affect its normal physiology. However, more research is needed to clarify various aspects of neurobrucellosis.


Introduction
Brucella spp.within the Order Rhizobiales and Class Alphaproteobacteria, are considered one of the most common zoonoses in the world and harmful intracellular bacteria.Brucella infects a wide range of wild and domesticated mammalian species.Brucellosis predominantly affects the reproductive system of animals, and chronic infection can disturb many organs (Hull and Schumaker, 2018;von Bargen et al., 2012).Several Brucella species can infect people using infected animal products (Pappas et al., 2006).Human brucellosis typically manifests as flu-like symptoms, such as an undulating fever, but it can also be a persistent infection that harms many organs (de Figueiredo et al., 2015;Dean et al., 2012).For the most part, Brucella spp.avoid the host immune system by adapting to the harsh intracellular environment and creating a replicative niche within the host's primary immune cells (dendritic cells and macrophages) (Roop et al., 2009;He, 2012).Brucella spp. is a pathogen that lacks many classical virulence characteristics.Small regulatory RNAs (sRNAs) are understudied Brucella virulence factors that post-transcriptionally modify gene function quickly in response to changing environmental variables (Waters and Storz, 2009).It was shown that the sRNAs AbcR1 and AbcR2 (AbcR1/2) predominantly serve as negative regulators of some ABC-type transport systems in B. abortus (Caswell et al., 2012) while describing the function of the sRNAs in B.abortus pathogenesis (Budnick et al., 2020).
Neuro-brucellosis, an inflammatory condition, is brought on by Brucella bacteria invading the nervous system.It mainly impacts the central nervous system (CNS), making the prognosis bleak (Pappas et al., 2006).Meningoencephalitis, demyelinating syndromes, brain abscesses, meningovascular disease, and myelitis are some neuro-brucellosis symptoms (Bouza et al., 1987;Young, 1989).Lesions are thought to be caused by the bacterium within the brain parenchyma, the pathological activity of inflammatory cytokines, and/or the demyelinating effect of an immunopathological response.CNS involvement typically occurs through hematogenous dissemination.Pleocytosis (the presence of leukocytes in the cerebrospinal fluid), one of the pathognomonic symptoms of neuro-brucellosis, is used to narrow down the differential diagnosis of this type of the illness together with the distinct production of intrathecal Abs (Baldi et al., 1999;Alba et al., 1992).
Brucellosis has a general clinical picture.Patients may experience an intense fever and other symptoms like general discomfort, exhaustion, weakness, headaches, and fatigue.The chronic phase, which includes relapses marked by fever, weakness, and diaphoresis, may follow the acute stage (Franco et al., 2007).Additionally, chronic brucellosis can cause neurological (Adeva-Bartolomé et al., 2005), musculoskeletal (Buzgan et al., 2010), mental (Maldonado-García et al., 2021), and osteoarticular problems.Anxiety and depression can show in brucellosis patients even when there is no apparent brain infection, suggesting that neuropsychological symptoms may result from the peripheral inflammatory response developed against the disease (Himmerich et al., 2019).Although there is no evidence to support this assumption, data demonstrating a link between IL-6 (a proinflammatory cytokine) blood concentrations and the emergence of anxiety and depression in infected patients (Naderi, 2016) are likely to support it.Additionally, it has been demonstrated that the intraperitoneal infusion of proinflammatory cytokines (IL-1, TNF-, and IL-6) either alone or in combination temporally promotes anhedonia and that mycobacterial infection may also result in memory problems and anhedonia (Lara-Espinosa et al., 2020).
There are still unresolved questions about the processes underlying these relationships, particularly those involving Brucella infections.However, published evidence suggests that they might include alterations in neurotransmitter availability in significant brain regions (Maldonado-García et al., 2021).
In light of this, the current study aims to assess the precise correlations between neuroinflammatory and cell death parameters in the brain's hippocampus.This is in Albino Wistar male rats exposed to Brucella abortus infection compared to the control group.

Animals
Twenty-four male albino rats, weighing 200-220 g, were acquired from our university's animal facility.Our research received approval from the university's Animal Care and Use Committee (IR.SBMU.AEC.1401.051).The rats were raised and housed in a controlled 12-hour light/dark cycle at 22 • C.They were then randomly assigned to either the control group or the Brucella group (12 rats in each group).

Bacterial culture
B. abortus 544 was cultured overnight in Brucella broth with constant stirring at 37 • C. Following 72 hours, the bacteria were collected through centrifugation at 5000 × g for 20 minutes at 4 • C and then rinsed in sterile phosphate-buffered saline (PBS).The bacterial cell concentration in the culture was approximated by comparing optical densities at 600 nm with an in-house standard curve.Inocula were prepared for intraperitoneal administration using sterile PBS.Bacterial cell concentration was assessed by plating cells on Brucella Agar.Each rat received an inoculation of 1 × 10 6 CFU (colony-forming units).

Stereological study; estimation of numerical density of neuron
After 14 days from the infection day, Stereological study was done.Rats were given deep anesthesia and then perfused with saline and 4 % paraformaldehyde in 0.1 M PBS through the heart.Following this, the brains of the rats were removed and placed in formalin for slide preparation.The hippocampal tissue samples from the rats were fixed in 4 % paraformaldehyde (PFA) for one week.These samples were then embedded in paraffin blocks and sliced longitudinally into ten μm thick sections using a microtome.To conduct a microscopic descriptive analysis of each group, the slides were stained with hematoxylin and eosin (H&E).Every ten sections were sampled, starting with a random number between 1 and 10.Subsequently, approximately ten sections from each rat were methodically randomized through uniform sampling.An unbiased counting frame was overlaid on the images, and a microcator was attached to the microscope's stage to measure the z-axis direction.The dissector height was set as a guard zone at the top and bottom of each section.Any neuron nucleus that came into maximal focus and was inside the counting frame without touching the exclusion line was selected.The numerical density of neurons in the hippocampus was estimated using the optical dissector method.Unbiased stereological estimations of the numerical density of CA1 neurons in the hippocampus were carried out.

Immunohistochemistry
14 days after infection, the rats were transcardially perfused with chilled saline and fixed using a fixator containing 4 % PFA in 0.1 M PBS after deep anesthesia using 100 mg/kg ketamine and 10 mg/kg xylazine.The brain was then removed, placed in PFA, prepared, and mounted on slides.The PFA-fixed tissues were embedded in paraffin wax.Three samples were chosen randomly from each group.The tissue section embedded in paraffin was usually cut using a rotary microtome (Thermo Fisher Scientific, Waltham, MA, USA) to a thickness of 6 μm.Each sample involved randomly selecting 8-10 coronal sections, followed by capturing five fields at high magnification.The slide was deparaffinized in xylene, passed through a series of diluted alcohols (100 %, 95 %, 70 %, and 50 %), and finally to water before staining.Antigen retrieval was performed before immunohistochemistry using 0.01 M citrate buffer solution (pH 6.0), 0.01 M PBS buffer (pH 7.0), 0.05 M EDTA (pH 8.0), 0.05 M Tris-EDTA (pH 9.0), and 0.05 M Tris-HCl.The slides were blocked in 10 % normal serum and 1 % BSA in TBS for one hour at room temperature.The primary antibodies used were rabbit polyclonal anti-GFAP antibody (ab7260, 1:500), rabbit monoclonal anti-Iba-1 (EPR16588, 1:500), rabbit monoclonal anti-caspase-3(EPR18297, 1:300), and rabbit monoclonal anti-Ki67(ab16677, 1:200), and they were diluted in PBS solution containing 0.3 % Triton X-100 and 1 % bovine serum albumin (BSA).
The sections were incubated in primary antibodies overnight at 4 • C. The sections were washed three times in PBS.They were consequently incubated with secondary antibodies to an avidin-biotin complex substrate in 0.05 M Tris buffer (pH 7.6) containing 0.05 % 3,3-diaminobenzidine tetrahydrochloride and 0.03 % hydrogen peroxide or secondary fluorescent FITC-conjugated antibodies.After the immunohistochemical reaction, the sections were washed three times with PBS and mounted in a Mounting Medium for IHC (Abcam, Cambridge, UK).Fluorescence microphotographs were captured by fluorescent microscopy (E200, Nikon, Japan).All the steps were performed by a researcher blinded to experimental conditions.

Glial morphology and distribution
Astrocyte and microglial cells were specifically labeled using GFAP and Iba-1 antibodies, respectively.Thirty individual cells that were GFAP+ or Iba-1+ were chosen and imaged using the 40X objective lens for reconstruction (Langhammer et al., 2010).The figure was imported into ImageJ (Java, NIH, USA).The nucleus of each microglia and astrocyte was marked in the center.Sholl analysis was performed to quantify the total process length based on previous instructions (Boroujeni et al., 2021a(Boroujeni et al., , 2021b;;Moghaddam et al., 2021).Each image was brought into ImageJ to measure the soma size.Using the Wand tools, the image scale was set, and then the border of each soma was chosen to calculate the nearest neighbor distance (NND).Y. Mao's Fiji (ImageJ) script was used to accomplish this .was used as described in previous studies (Davis et al., 2017a(Davis et al., , 2017b)).The regularity index (RI) was calculated as follows: where X NND is the average NND of a population, and δ NND is the standard deviation of that population (Davis et al., 2017b;Wäussle et al., 1993).
The arbor area was calculated by manually creating a polygon that connected the endpoints of the appendages using the ImageJ Polygon tools (Davis et al., 2017a(Davis et al., , 2017b)).

Western blotting
After 14 days from the infection day, hippocampus tissues were collected after inducing deep anesthesia with 100 mg/kg ketamine and 10 mg/kg xylazine and making use of a decapitation guillotine.The tissues were then lysed for 2 minutes on ice in lysis buffer (Tris-HCl, sodium deoxycholate, sodium dodecyl sulfate (SDS), NaCl, EDTA, Triton X-100, complete protease inhibitor cocktail).The samples were then cleaned by centrifugation at 12,000 rpm for 30 minutes at four • C. Bradford technique was used to measure the protein concentration.Then, 60 µg of total proteins were electrophoretically separated on SDSpolyacrylamide gels and transferred to polyvinylidene difluoride membranes.For 1 hour, the blots were then blocked with a 2 % skimmed milk-blocking solution.The membranes were treated overnight at four • C with a primary antibody against caspase-3.After 60 minutes of incubation with the secondary antibody at room temperature, immune reactive bands were detected using an ECL kit.They were captured on Kodak X-ray films.Densitometric data for protein bands were acquired with ImageJ.

Statistics
All statistical analyses were conducted in SPSS version 23.The graphs were created with Graph Pad Prism 7. The collected data are presented as the mean ± SEM.The independent samples t-test was used to determine the differences between the experimental groups.P val-ues<0.05were considered significant.

Brucella increases hippocampus astrogliosis
After 14 days from the infection day, and using a specific marker for astrocytes (GFAP), IHC was performed to detect astrocytes and any astrogliosis in the hippocampus of the subjects (Fig. 1a).As shown in Fig. 1b, the number of GFAP + cells were higher in Brucella than the control group (P<0.001).Moreover, Brucella decreased NND and regularity index compared to the control (P<0.001,P<0.01, respectively) (Fig. 1c, d).The results showed that Brucella increases the proliferation of astrocytes in the hippocampus.

Brucella infection changes the morphological characteristics of astrocytes in the hippocampus
After 14 days from the infection day, the results showed that morphological complexity (Fig. 2a) and total astrocyte process length (Fig. 2b) were significantly reduced in the Brucella group compared to the control group; however, the soma size was significantly larger than in the control group (Fig. 2c).Rats infected with Brucella showed markedly reduced astrocyte soma roundness (Fig. 2d) and arborization area (Fig. 2e) compared to control rats.However, the percentage of the primary branch of astrocytes in the control group and Brucella group were 51.9 % and 67.7 %, respectively.The percentage of secondary branches in the control and Brucella groups was 40.4 % and 31.3 %, respectively.The tertiary arbores of astrocytes in the control and Brucella groups were 19.2 % and 15.6 %, respectively (Fig. 2f).Our results indicated that Brucella changes the morphology of astrocytes and activates astrocytes in the hippocampus of rats.

Brucella Increases microgliosis in rats' hippocampus
Specific staining was used to identify and examine microglia and any microgliosis in the hippocampus regions of subjects infected with and not infected with the Brucella.Iba-1 microglia marker IHC staining (Fig. 3a).The quantity of Iba-1 positive cells was counted to compare the two groups (Fig. 3b).Both the regularity index and NND significantly decreased in Brucella-infected rat hippocampal samples compared to the control (Fig. 3c and d).The results indicated that Brucella increases the proliferation and migration of microglia in the hippocampus.

Brucella-infected
The Sholl analysis showed that Brucella-infected animals significantly reduced microglia complexity compared to the control group (P < 0.001) (Fig. 4a).Our results showed that the total microglia process length was decreased in the Brucella group compared with the control group (P < 0.01) (Fig. 4b).Moreover, Brucella-infected rats increased microglia soma size compared to the control group (P < 0.001) (Fig. 4c).Moreover, the soma roundness and microglia arbor area decreased in Brucella-infected rats compared to the control group (Fig. 4d, e).However, the percentage of primary branches of microglia in the control and Brucella groups was 39.4 %, 77.7 %, and 86.5 %, respectively.Also, the percentage of secondary microglia branches in the control and Brucella groups was 49 % and 12.5 %, respectively.The tertiary arbores of microglia in the control and Brucella groups were 11.5 % and 8.3 %, respectively (Fig. 4f).The results showed that Brucella changes the morphology of microglia and activate of microglia in the hippocampus of rats.

Brucella infection induces the expression of caspase-3 in hippocampal cells
After 14 days from the infection day, the effect of Brucella inoculation on caspase-3 expression as assessed by IHC and western blotting.Rats given Brucella-infected rats showed higher levels of caspase-3 expression in their western blotting results when compared to the control group (Fig. 5a).On the other hand, IHC showed caspase-3 expression increased in the Brucella-infected group compared to the control group (Fig. 5b).Our results showed that Brucella increases the expression of apoptotic factor caspase-3 in the hippocampus of rats.

Brucella infection decreases Ki67 expression in the hippocampus
For cell proliferation detection, IHC measured the neurogenesis factor Ki67. Comparing the Brucella-infected rats' hippocampal tissue to that of the control rats, the results showed a significant decrease in Ki67 expression in the dentate gyrus (Fig. 6).

Brucella infection increases dark neurons
Our results showed that Brucella infection reduced the number of pyramidal neurons in the hippocampus compared to the control group, but this was not statistically significant (Fig. 7a, b).Moreover, the mean number of dark neurons increased in the Brucella group than in the control group (P<0.001) (Fig. 7c).Consistent with the neuronal cell counts, the results showed a remarkable decrease in soma diameter in the hippocampal pyramidal neurons in the Brucella-infected group compared to the control group (P<0.001) (Fig. 7d).

Discussion
Neurobrucellosis is a significant challenge in infectious diseases due to its diverse clinical symptoms (Gul et al., 2009;Soares et al., 2022).Brucellosis is an inflammatory condition.Inflammation observed in the disease's acute and chronic stages, affecting various organs, including the brain.To elucidate the underlying cause of the inflammatory state associated with neurobrucellosis, the present study was conducted to examine the specific relationships between astrogliosis/microgliosis and apoptosis/neurogenesis in the hippocampus of This paper presents empirical findings suggesting that the B. abortus infection and its inflammatory response can impact the brain without attempting to detect the microorganism in the brain.This impact involves astrogliosis and microgliosis in adult male rats' hippocampus.An increasing body of evidence suggests that inflammation may play a significant role in the pathogenesis of various CNS disorders.Astrocytes and microglia play crucial roles in the innate immune response of the CNS when triggered during pathological situations such as microbial infections, including brucellosis (Esen et al., 2004;García Samartino et al., 2010;Rodríguez et al., 2017Rodríguez et al., , 2024;;Miraglia et al., 2016Miraglia et al., , 2013Miraglia et al., , 2018) ) and neurodegenerative disorders (Perry, 2004;Rock et al., 2005;Vohra et al., 2002).The infection of microglia and astrocytes by B. abortus results in the production of several cytokines and chemokines.While these proinflammatory mediators could contribute to the host's defense against B. abortus, they may also significantly impact the start and progression of inflammatory and innate immune responses in the brain (García Samartino et al., 2010).
Our stereology investigation indicates that Brucella infection leads to a notable augmentation in astroglial growth.Moreover, it has been observed that the length of astrocytic processes is reduced in the Brucella group.However, the cellular body within astrocytes linked with Brucella infection undergoes enlargement.These observations provide evidence that Brucella infection not only increases the number of astroglial cells but also causes changes in the physical structure of astrocytes.This triggers a reactive response in these cells, all leading to astrogliosis.Astrogliosis is the non-specific expression of many pathological brain conditions.Astrocytes that undergo gliosis have augmented cellular volume, enhanced proliferation rate, and active expression of specific proteins (Ridet et al., 1997;Mucke and Eddleston, 1993).
Various types of injury, including infections (e.g., HIV encephalitis and Lyme neuroborreliosis), tumors, autoimmune disorders, or ischemia, can trigger gliosis.Astrogliosis was also observed in neurobrucellosis (Seidel et al., 2003;Sohn et al., 2003).In this study, we have provided further evidence that Brucella infection in the hippocampus of rats without prior exposure induces astrogliosis.Both morphological and immunohistochemical alterations demonstrate this.
Activated microglia secrete several neurotoxins, such as proinflammatory cytokines and NO (Liu et al., 2002).While NO is a significant neuromodulator at normal concentrations, increased amounts contribute to neuropathogenesis.The elevation in NO levels and superoxide leads to an increase in the production of peroxynitrite anions, which possess high toxicity (Bartesaghi and Radi, 2018).Peroxynitrite activation can induce several pathological problems, all leading to metabolic dysfunction, DNA damage, and, finally, cell death.In line with this evidence, our immunohistochemical and western blot analyses targeting caspase 3, an apoptosis marker, reveal a notable elevation in its expression within the Brucella group.This observation suggests apoptosis and cellular demise following Brucella exposure.Taken together, the interaction and mutual reinforcement between neuroinflammation and oxidative and nitrosative stress can have a role in both the development and persistence of neurobrucellosis (He et al., 2020).Due to Moley et al. study, genes linked to interferon responses and inflammation were significantly up-regulated  in Brucella-infected brains, while genes related to neurologic function were significantly down-regulated, according to transcriptional analysis.Brucella colonized the brains of mice lacking type II interferon signaling, but mice lacking both type I and type II interferon signaling developed clinical signs of neurobrucellosis more quickly, showed loss of hippocampal neurons, and had higher concentrations of Brucella in their brains than mice lacking type II interferon signaling alone (Moley et al., 2023).
Our stereology investigations revealed a notable augmentation in the number of dark neurons within the Brucella group.dark neurons are frequently observed in clinical and experimental neuropathologic conditions (Ahmadpour et al., 2019).As per Atillo et al. findings, it has been observed that dark neurons display a compacted dark staining pattern, accompanied by an elevated occurrence of inflated mitochondria (Atillo et al., 1983).Before the 1980s, the prevailing belief only supported that dark neuron formation was caused by artifactual factors outside neurons (related to fixation and staining processes).However, recent experimental studies have identified four distinct types of dark neurons: artefactual, neostriatal (associated with Huntington's disease), irreversible, and reversible types (Ahmadpour et al., 2019;Ahmadpour and Haghir, 2011).Although a complete understanding of the manner of death and pathogenesis of dark neurons has yet to be achieved (Gallyas et al., 2008;Kövesdi et al., 2007), dark neurons have been observed in pathological situations, such as ischemia, epilepsy, depression, and hypoglycemia (Catarzi et al., 2007).In addition, dark neuron development has been shown in conditions characterized by high-stress levels, such as severe physical stress, and in the natural process of aging in the cerebellum (Vohra et al., 2002;Ishida et al., 1999).All of these pathological disorders result in disruptions to an elevation in excitatory neurotransmitters (e.g., glutamate), ion gradients, and the formation of free radicals (Vohra et al., 2002;Kherani and Auer, 2008).
There is a prevailing belief that heightened levels of cytokines and NO have a detrimental impact on neurogenesis regulation in the brain.In our previous study, we observed that COVID-19 infection, which is also known to overstimulate the immune and inflammatory responses, the viral infection led to a decrease in neurogenesis, as depicted by the significantly decreased level of Ki-67 (Bayat et al., 2022).In this work, we also observed a reduction in neurogenesis, as indicated by Ki-67 staining, in the dentate gyrus of the hippocampus in rats infected with B. abortus.This finding shows that a decrease in neurogenesis could serve as a pathological mechanism in neurobrucellosis.The primary cause of reduced neurogenesis is likely a decline in brain-derived neurotrophic factor (BDNF), as observed in previous studies (Chen et al., 2008).However, He et al (He et al., 2020).reported contrasting findings.Their study established a mouse model of chronic fatigue syndrome (CFS) by subjecting mice to six consecutive injections of antigens from heat-killed B. abortus at two-week intervals over 12 weeks.The researchers assessed hippocampal neurogenesis using Ki-67, doublecortin (DCX), and 5-bromodeoxyuridine (BrdU) tests.Their findings revealed no significant reduction in neurogenesis inside the hippocampus of mice with CFS.Further investigation is required to elicit additional information regarding this controversy in future studies; however, different modeling techniques and dosage and duration of exposure to B. abortus seem to be to blame.

Conclusions
The current study demonstrated the pathogen's capacity to destroy the hippocampus and potentially its normal physiology; however, more research is required to clarify various aspects of neurobrucellosis.

Fig. 1 .
Fig. 1.Brucella induces astrogliosis in hippocampus of infected rats.As illustrated in the histological images, Brucella-infection increased the number of astrocytes following the specific staining of GFAP.The first graph (a) quantitively verified this increasing in the number of hippocampal astrocytes.As indicated in c and d, quantitative indicators of the nearest neighbor distance of astrocytes, NND, and regularity index in the Brucella group diminished significantly (*P < 0.05; **P < 0.01; ***P < 0.001).The values were expressed as means ± SEM.

Fig. 2 .
Fig. 2. Brucella induces morphological changes in hippocampal astrocytes of infected rats.The astrocytes' overall complexity dropped in the infected animals' hippocampus (a).Though the Brucella infection reduced the complexity of astrocyte processes compared to the control (b), an increase in the soma size was observed in the Brucella group (c).Also, the Brucella group had a significant fall in the soma roundness (d) and arborization area (e) compared to the control.Brucella infection reduced the percentage of secondary and tertiary astrocytes' arbors but increased the percentage of primary arbors of astrocytes (f) (*P<0.05;**P<0.01;***P<0.001).The values were expressed as means ± SEM.

Fig. 3 .
Fig. 3. Brucella induces microgliosis in the hippocampus.Regarding (a), which includes immunohistochemical staining for the microglial marker (Iba-1), there is a considerable increase in the number of Iba-1 positive cells in the hippocampus (an indicator of microgliosis in the Brucella group) (b).Also, two quantitative indicators of the nearest neighbor distance of microglia, NND (c) and regularity index (d), were shown to diminish in the Brucella group compared to the control (*P < 0.05; **P < 0.01; ***P < 0.001).The values were expressed as means ± SEM.

Fig. 4 .
Fig. 4. Brucella activates the microglia in the rat's hippocampus.include various results: Brucella contamination reduced the complexity of the glial cells (a).As a result of Brucella infection, there is a drop in the total microglial process length (b), soma size (c), soma roundness (d), and microglia arbors area (e).Also, in contrast to the drop in the percentage of secondary and tertiary arbors of microglia in microglia of the hippocampus in Brucella-infected rats, the percentage of the primary arbor went up in this type of glial cells (f).(*P<0.05;**P<0.01;***P<0.001).The values were expressed as means ± SEM.

Fig. 5 .
Fig. 5. Brucella induces the expression of caspase-3 in the hippocampus.Western blot data showed that hippocampal caspase-3 expression raised following the infection (a).As illustrated in the images and graphs, the result of merging the photos related to the DAPI and Caspase-3 proved a significant increase in caspase-3 expression in hippocampal cells in the Brucella group relative to the control (b) (**P<0.01;***P < 0.001).The values were expressed as means ± SEM.

Fig. 6 .
Fig. 6.Brucella decreases the expression of Ki67 in the hippocampus.Markedly, there was a drop in the cell population in the hippocampal tissue in Brucella compared to the control.Both the histological section and graph prove that Brucella infection in the brain leads to a fall in the neurogenesis in the hippocampus (**P<0.01;***P < 0.001).The values were expressed as means ± SEM.

Fig. 7 .
Fig. 7. Brucella increases the number of dark neurons in the hippocampus.The graphs demonstrate that while there is no significant effect on the total number of neurons (a), the Brucella infection significantly increases the number of dark neurons (b) and reduces soma diameter (c).(*P<0.05;**P<0.01;***P<0.001).The values were expressed as means ± SD.