Preparation of oil extract
Rosemary (R. officinalis L.) essential oil used in this work was kindly extracted by the Department of Botany, Faculty of Science, Mansoura University (Mansoura, Egypt). The oil extraction was performed using hydrodistillation process following the described protocol by Tigrine-et al,(Tigrine-Kordjani et al. 2012) and the oil was kept at room temperature till be used. As described by manufacturer, the rosemary oil contained about 32.5% 1,8-cineole, 13.7% α-pinene, 11.3% β-pinene 15.2% p-cymene, 12.2% camphene, 8.6% camphor and 6% of other unidentified compounds.
Animals
Fifty adult male rats (Sprague Dawley strain, 3 months old and 200–250g weight) were used in this study. They were housed in suitable cages (5 rats/cage) under standard hygienic environmental conditions (12h light/12h dark cycle, 25±5oC, and 65% humidity) and freely supplied with a balanced diet and clean water. The protocol of this experiment was performed according to the ethical committee Guidelines of Faculty of Veterinary Medicine, Mansoura University (Approval number; R/4/307/2019).
Study design
The rats were randomly divided into five groups (10 rats /group) as follows: Group I: control group; no manipulation was received. Group II: IBO–model group; bilaterally intra-hippocampus injected with IBO acid (4 μL). Group III: cell-only group; bilaterally intra-hippocampus injected with IBO acid (4 μL) followed by bilateral intra-hippocampus transplantation of hOBNSCs (4 μL) 22days after IBO acid injection. Group IV: cell + rosemary oil (ROO) before transplantation; injected of IBO acid as in the 2nd group, followed by rosemary oil administration (30mg/kg) by stomach tube at on the 7th day and once a day thereafter for 2; this was then followed with hOBNSCs transplantation on the 22nd day as in the third group. Group V: cells with rosemary oil (ROO) concurrently at transplantation; injected the IBO as in the 2nd group 22 days after hOBNSCs transplantation as in group III and rosemary oil administration (30mg/kg) once a day for 2 weeks
Human olfactory bulb neural stem cells (hOBNSCs) isolation and culture
The hOBNSC were isolated and cultured according to the methods described by Casalbore et al(Casalbore et al. 2009).This step was explained in detail in our previous studies (Marei et al. 2015a)(Marei et al. 2015b) (Marei et al. 2016). The olfactory bulbs were collected from six adult patients (39-45 years) undergoing craniotomy at the Institute of Neurosurgery, Catholic University, Rome, Italy. Informed consent was received according to the Ethical Committee protocols of the Catholic University.
Immunocytochemical assessment
Immunostaining of the cells’ composition was performed with appropriate specific antibodies to evaluate the cells’ multipotentiality, using the method published by Pagano et al., (Pagano et al. 2000). The primary antibodies used (all from Sigma-Aldrich) were anti-Nestin (1:200, rabbit, for undifferentiated neural stem cells), anti-beta tubulin ІІІ (1:100, rabbit, for immature neurons), anti-MAP2 (1:200, rabbit, for mature neurons), anti-GFAP (1:400, mouse, for astrocytes) and anti NG2 (1:100, rabbit, for oligodendrocyte progenitors). The secondary antibodies (also from Sigma-Aldrich) were tetramethylrhodamine isothiocyanate [(TRITC), 1:200, anti-rabbit, anti-mouse, and Fluorescein iso-thiocyanate (FITIC), 1:200, anti-rabbit, anti-mouse. After that, the culture was washed and incubated for 15 min with 4, 6-diamidino2 phenyl indole dihydrochloride [(DAPI), nuclear stain, 1mg/ml, and then examined with FluorsaveTM [Calbiochem; La Jolla, CA, USA].
Morris water maze test (MWMt)
After acclimatization to the environment for one week, the rats were subjected to the Morris water maze to assess spatial memory and learning. The methodology of this test was completely explained by (Vorhees and Williams 2006). The animal’s behavior, the escape latency time (time needed to reach to platform), and time in the target quadrant (quadrant time) were tracked and measured with a digital camera. The scores were recorded on the 6th day after training (before IBO injection) and at the end of the study (before sample collecting with one week).
IBO acid-induced memory deficient rat model
Memory deficient model was performed by bilateral IBO acid (Sigma, St. Louis, MO) injection into the rat’s hippocampus. IBO acid was dissolved in 10mM artificial cerebrospinal fluid (CSF) at a concentration of 8 mg/ml (Karthick et al. 2016b). The surgery protocol was the same as that explained in our previous study (Marei et al. 2015a). To anesthetize the rats, a mixture of ketamine (80mg/kg body weight) and Xylazine (10mg/kg body weight) was intraperitoneally administered. Each rat was carefully placed in a stereotactic frame under complete aseptic conditions. Intra-hippocampus injection of 4 μL of IBO acid solution was made slowly over 5 minutes using a 10 μL Hamilton syringe (coordinate, ML: 2.5 mm, AP: 3.5 mm, and VD: 2.7 mm relative to the bregma) according to the brain atlas of Paxinos and Watson (Paxinos and Franklin 2001). Following the injection, the skin was sutured, and the rats were returned to their cages when fully recovered from anesthesia.
hOBNSCs transplantation
Animals in groups III, IV, and V were subjected to hOBNSCs transplantation surgery on the 22nd day after IBO injection. A Trypan blue exclusion test was used immediately before cell transplantation to detect cell viability (not less than 95 % viability) under a phase-contrast microscope(Glass et al. 2012). The total cell number was calculated with a hemocytometer then the cells were suspended in artificial CSF (60,000 cells/µL). Animals were re-anesthetized and 4 µL of cell suspension was transplanted (2 µL for each side) into each rat using a 10 μL Hamilton syringe at the same coordinates mentioned above. The immune suppressor cyclosporine (Sandimmune®) was injected daily (10 mg/kg/S.C.) one day before cell grafting and throughout the experiment’s course.
Sample collection
Samples were concurrently collected from each group 11 weeks after IBO acid injection (8 weeks after cell transplantation). Blood samples were collected through cardiac puncture and divided into 2 parts. The first part was collected into EDTA tubes for total leukocyte count (TLC) and differential leukocyte count (DLC). The second part was collected into plain clean centrifuge tubes and allowed to clot at room temperature for 30 min. The serum was separated by centrifugation at 3500 r. p.m. for 10 min then aspirated by automatic pipette and kept frozen at −80 °C for further estimation of cytokine levels. After that, rats were euthanized, and their brains were dissected. The hippocampus was collected from each rat. Tissue samples were divided into three groups. The 1st group was fixed in neutral formalin 10% (48hrs) for histological examination and morphometric analyses; the 2nd group was preserved in Trizol Reagent (Invitrogen, UK) for gene expression analyses and the 3rd group was homogenized and cold centrifuged; the supernatants were separated and carefully collected into clean sterile tubes to be used in the evaluation of antioxidants and oxidative stress parameters.
Total leukocyte and differential leukocyte count
Blood samples were diluted with Turks' solution (1:20) for manual calculation of TLC under a light microscope (10x lens) using a hemocytometer according to the method of Wintrobe, 1993 As well, a thin blood film was spread on a glass slide, air-dried and fixed in absolute methanol (100%). The dried slides were stained with Giemsa stain. Later, 200 leukocytes cell on each slide were examined under an oil immersion lens (100 x) and used to determine the absolute count of normal neutrophils, toxic neutrophils, total neutrophils, lymphocytes, and monocytes. Neutrophil lymphocyte ratio (NLR) and lymphocyte monocyte ratio (LMR) were calculated according to ([CSL STYLE ERROR: reference with no printed form.]).
Serum cytokines and C-reactive proteins (CRP)
Serum tumor necrosis factor-alpha (TNF-α) and IL-1β concentrations were measured in serum using ELISA, with ready-made commercial kits purchased from Quantikine Co. (USA). CRP was estimated according to the standard protocol of ELISA ready-made kits obtained from Cobas Co. (USA).
Antioxidants and Oxidative stress parameters
Catalase (CAT) activity was assessed in hippocampus tissues according to the method of Claiborne (Greenwald 1985), while glutathione (GSH) level was determined according to the method of (Jollow et al. 1974) and superoxide dismutase activity (SOD) was measured according to (MARKLUND and MARKLUND 1974).Furthermore, Malondialdehyde (MDA) level was determined according to the technique of (Todorova et al. 2005).Total Nitric oxide (NO) levels were converted to stable nitrite/nitrate and determined using Greiss’ reagent according to the method of (Miranda et al. 2001) with the modification of using zinc sulfate for protein precipitation.All other parameters were determined in hippocampus tissue homogenates using a spectrophotometer (Lambda EZ201; Perkin Elmer).
Total RNA extraction and gene expression analysis
RNA was totally extracted from the hippocampus specimens with the use of Trizol Reagent (Invitrogen, UK) following the procedures of the manufacturer. The concentration of RNA and purity were checked with a “Q5000” Quawell nanodrop spectrophotometer (USA). The integrity of RNA was also assessed by gel electrophoresis. An equivalent of 2mg of RNA was transferred to cDNA with a kit supplied from Intronbio, South Korea (HisenscriptTM cDNA synthesis kit) according to the instructional manual.
Quantitative real-time PCR (qRT-PCR) was performed on a Rotor-Gene Q cycler (Qiagen, Heidelberg, Germany), using QuantiTect SYBR Green PCR kits (Qiagen, Heidelberg, Germany). The sequences of the primers used in this study are shown in Table 1. The ß-actin gene acted as an internal control for normalizing expression levels of the target genes(Livak and Schmittgen 2001). The total volume of the reaction is 20 µL, which contained 10 µL 2x SensiFast SYBR, 3 µL cDNA, 5.4 µl for H2) (distilled water), and finally 0.8 µl of each gene-specific primer. The amplification cycling conditions were: 95 oC for 10 min then 40 cycles of 94 oC for 40 sec; 55 oC for 30 sec is the optimum temperature for annealing of the chosen primers; then elongation for 30 sec at a temperature of 72 oC and again at 72 oC for final elongation over 10 min. At the end of the amplification cycle, a melting curve was created after completion of the amplification phase; the relative expression analysis of target genes was complete using the 2-ΔΔCt procedures (Pfaffl 2001).
Histomorphometric analyses
Formalin-fixed hippocampus samples were processed for paraffin wax embedding by dehydration in ascending grades of ethanol (50, 70, 80.95, 100,100/1hr for each), then cleared in two changes of xylene (1hr/each); they were paraffin wax impregnated and embedded, sectioned (5 μm) with a rotatory microtome and mounted on coated glass slides. The mounted sections were stained with Hematoxylin & Eosin (H&E) stain or cresyl violet stain and examined under a light microscope. For morphometric analysis, morphometric measures were obtained from all groups. Five different samples from five different rats were taken. Three H&E-stained sections from each sample were examined under higher magnification (x400)(Gao et al. 2006). The mean thickness of the pyramidal layer at CA1 and CA3 and the granular layer of the dentate gyrus was measured with an image analyzer. At the same time, the mean number of their viable neurons was also counted.
Statistical analysis
Statistical analysis of the results was performed using SPSS PC (version 19, IBM Analytics, New York, New York, USA). All values were expressed as mean ± SE. The data were analyzed using a one-way analysis of variance (ANOVA) test followed by Tukey's post hoc test for multiple comparisons. Differences were considered statistically significant at p <0.05.