The effect of intra-nasal co-treatment with insulin and growth factor-rich serum on behavioral defects, hippocampus histological, and oxidative-nitrosative stress changes induced by icv-STZ in a rat model

doi:10.21203/rs.3.rs-3238680/v1

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The effect of intra-nasal co-treatment with insulin and growth factor-rich serum on behavioral defects, hippocampus histological, and oxidative-nitrosative stress changes induced by icv-STZ in a rat model

https://doi.org/10.21203/rs.3.rs-3238680/v1

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Impaired insulin and growth factor functions are thought to drive many alterations in neurodegenerative diseases like dementia and seem to contribute to oxidative stress and inflammatory responses. Recent studies revealed that nasal growth factor therapy could induce neuronal and oligodendroglia protection in rodent brain damage induction models. Impairment of several growth factor signaling was reported in neurodegenerative diseases. So, in the present study, we examine intranasal co-treatment of insulin and a pool of growth factor-rich serum (GFRS) which separated from activated platelets on memory and behavioral defects induced by intracerebroventricular streptozotocin (icv-STZ) rat model also investigate changes in the hippocampus oxidative-nitrosative state and histology. We found that icv-STZ injection (3 mg/kg bilaterally) impairs spatial learning and memory in Morris Water Maze, leads to anxiogenic-like behavior in the open field arena, and induces oxidative-nitrosative stress, neuroinflammation, and neuronal/oligodendroglia death in the hippocampus. GFRS (1µl/kg, each other day, 9 doses) and regular insulin (4 U/40 µl, daily, 18 doses) treatments improved learning, memory, and anxiogenic behaviors. The present study showed that co-treatment (GFRS + insulin with respective dose) has more robust protection against hippocampus oxidative-nitrosative stress, neuroinflammation, and neuronal/oligodendroglia survival in comparison with the single therapy. Memory and behavioral improvements in the co-treatment of insulin and GFRS could be attributed to their effects in the reduction of oxidative stress and neuroinflammation in the hippocampus.

Neurotoxicity

Hippocampus

Oxidative stress

Growth factor

Memory

Anxiety

Epidemiological reports consider Alzheimer’s disease as the most common type of dementia. Difficulty in remembering recent events is an early sign of dementia. As the disorder progresses, many symptoms develop, such as lack of space and time awareness, mood swings, confusion, and behavioral changes like depressive or anxiogenic behaviors (Winblad et al. 2016). Dementia, which begins after the age of 65 without a family history [elderly sporadic Alzheimer's disease (sAD)], is the most common type of Alzheimer's disease and has several causes and risk factors (Bettens et al. 2013).

Neuropsychiatric disorders, like anxiety, were observed in AD (Zhao et al. 2016). Studies have shown anxiogenic behavior increases in dementia induction models (España et al. 2010; Pamplona et al. 2010). Anxiety is related to sAD development, so it is considered a risk factor for AD. Some studies found that anxiety is associated with AD-related CSF markers in subjects with mild cognitive impairment (Ramakers et al. 2013). Studies found anxiety symptoms in amnestic mild cognitive impairment were attributed to medial temporal atrophy and concluded, anxiety accelerates AD progression (Mah et al. 2015; Pietrzak et al. 2015). Therefore, any relationship between anxiety and Alzheimer's and treatment strategy needs to be investigated (Becker et al. 2018).

Morphological changes in Alzheimer patients’ brains include enlarged brain ventricles and progressive medial temporal lobe atrophy, in which hippocampal atrophy occurs earlier than other areas of the brain (Henneman et al. 2009). Studies have shown that the hippocampus is very important for the formation of spatial memory (O'Keefe and Dostrovsky 1971).

Reasons associated with sAD have been not fully known and over time its number is increasing, but theories for the causes of the disease include accumulation of amyloid plaques, the formation of neurofibrillary tangles, cholinergic neuron activity reduction in the brain, neuroinflammation, mitochondrial disorders or oxidative-nitrosative imbalances of neurons which finally causes neuronal and synapse loss, especially in hippocampus and cortex. Therefore, sAD is considered a multifactorial disease (Barnes and Yaffe 2011).

Reports have shown a significant reduction in the antioxidant activity of superoxide dismutase (SOD) and catalase enzymes and lipid peroxidation elevation in the temporal lobe of Alzheimer's patients (Marcus et al. 1998). Oxidative-nitrosative-produced molecules from brain metabolism were important factors in AD pathogenesis and happened earlier than the amyloid plaques and neurofibrillary tangles deposit formations. It has been indicated that oxidative stress causes excitotoxicity which leads to the formation of hyperphosphorylated tau and in turn this defective protein triggers neuroinflammation. These immune reactions of inflammation disrupt the physiological function of glial cells, leading to the accumulation of amyloid-beta. Accumulation of this protein causes a defect in neurotransmitter secretion at synapses, and eventually, these disorders lead to cognitive deficits (Mehan et al. 2012; Moreira et al. 2009).

The oxidation appears to induce immune reactions, which ultimately disrupt a chain of mechanisms like growth factor regulation in Alzheimer's disease (Casadesus et al. 2007). Studies have shown that insulin signaling pathways are disrupted by neuroinflammation (Bedse et al. 2015b). Gene expression and phosphorylation of tau protein are regulated by the insulin signaling pathway, which shows a relationship between Alzheimer's pathology and insulin signaling impairment (Schubert et al. 2003).

Chronic activation of glial cells causes the continuous release of inflammatory mediators, and glial cells no longer secrete growth factors, induce cytotoxic effects, and cause the progression of Alzheimer's disease (Bouvier and Murai 2015). Microglia engaged in inflammation are considered a major source of reactive oxygen species in the brain (Bisht et al. 2018). Astroglia cells play an essential role in maintaining the blood-brain barrier (BBB) and studies report that BBB breakdown and dysfunction is a key factor in Alzheimer's disease progression (Altman and Rutledge 2010).

Icv-STZ injection causes progressive deficits in learning, memory, perception, and behavior, which is caused by brain energy metabolism impairment, mitochondrial dysfunction, neuroinflammation, and oxidative-nitrosative stress. So, it is used as a laboratory model to investigate the process and treatment of sporadic Alzheimer's disease in rat brains (Kamat et al. 2016). Icv-STZ injection leads to the loss of neuronal and oligodendroglia cells in the rat brain (Lester-Coll et al. 2006). Many molecular disorders caused by icv injection of STZ are related to impaired signaling pathways of insulin-like growth factor 1 (IGF-1) and insulin (Grieb 2016).

Insulin and growth factors pass the blood-brain barrier through selective and saturable receptor-dependent transmission, which is influenced by various factors such as morbid obesity, inflammation, diabetes mellitus, and triglyceride levels (Heni et al. 2014; Sartorius et al. 2015). To refrain from these limitations, intranasal treatment seems to have a better therapeutic outcome, and growth factors could bypass the blood-brain barrier and enter to CNS through the peri-vascular, olfactory, and trigeminal nerve pathways (Costantino et al. 2007).

Studies have shown that intranasal administration of insulin improves memory impairments, reduces neuroinflammation, and increases cAMP response element-binding protein (CREB) phosphorylation resulting in brain-derived neurotrophic factor (BDNF) level elevation in the hippocampus of STZ-induced Alzheimer's rats (Rajasekar et al. 2017).

It has been indicated that the levels of some growth factors such as nerve growth factor (NGF), IGF-1, vascular endothelial growth factor (VEGF), and BDNF are reduced in the brains of Alzheimer models (Houeland et al. 2010; Moloney et al. 2010; Nagahara et al. 2009). Growth factors increase intracellular cAMP which ultimately causes CREB phosphorylation and activation. CREB-derived factors are vital for many important functions of CNS including neurogenesis, neuronal survival, neuronal growth, and differentiation. It has been proven that activating the CREB signaling pathway is essential for memory consolidation (Alberini 2009).

Several studies have investigated the neuroprotective effects of intranasal administration of growth factors such as transforming growth factor-beta 1 (TGF-B1), fibroblast growth factor 2 (FGF-2), IGF-1, VEGF, BDNF, and insulin in various neurological disorders (Jin et al. 2003; Lochhead and Thorne 2012). However, most studies have focused on using one growth factor which may be an inadequate approach because, in neurodegeneration, many of the growth factor signaling are impaired, not one. An alternative way to growth factor therapy is the administration of a growth factors pool derived from plasma and platelets (Shen et al. 2009).

Platelet-rich plasma (PRP) is known as a biological product with high platelet concentrations that is prepared from the isolation of plasma. If the platelets are activated in the plasma, a large number of growth factors [such as platelet-derived growth factor (PDGF), IGF-1, VEGF, TGF-b, and FGF2] from the alpha granules of the accumulated platelets are secreted. After separating the jelly scaffold from the solution, PRP will be converted into GFRS which has a lot of therapeutic applications (Anitua et al. 2012).

Considering that many growth factors decrease in the brains of icv-STZ rats and platelets contain some of these growth factors and studies have also shown that platelet-derived growth factors and insulin have neuroprotective effects, therefore, this study aimed to examine the effects of co-treatment of intranasal administration of growth factor-rich serum and insulin as well as the interfering effects of them on memory and behavior defects induced by icv-STZ rat model, moreover, evaluate hippocampus volume, cells number and oxidative-nitrosative state changes.

2.1 Animals

The experiment was done with forty male Sprague-Dawley rats from a weight range of 210–240 g, purchased from the Comparative and Experimental Medical Center of the Shiraz University of Medical Sciences (SUMS). All rats had standard conditions (temperature: 22 ± 2 °c, relative humidity: 50%, with a 12-hour light/dark cycle) and had free access to laboratory food and water. The animals were habituated to handling for nasal administration for 14 days before the experiment. To prevent seasonal disturbances, experiments were started on all groups at the same time. Animal procedures were conducted in accordance with the National Institutes of Health’s Guide for Care and Use of Laboratory Animals and the Animal Research: Reporting in Vivo Experiments (ARRIVE) Guidelines and approved by the Ethics Committee of the university (approval number: IR.SUMS.REC.1398.168).

2.2 Experimental design

The rats were randomly divided into five groups (n = 8/group) including Control, STZ (streptozotocin), STZ- IN (STZ + insulin), STZ- GFRS, and STZ- IN + GFRS. Animals were kept in large polyacrylic cages (each cage contains one rat from each group). The control group received an icv injection of 10 µl saline 0.9% and other groups received an icv injection of 3 mg/kg STZ (Sigma, USA) dissolved in 10 µl sterile saline 0.9% STZ on days 1 and 3 of the experiment. The injection was done slowly into both lateral ventricles (each side 5µl). For nasal treatment, the control and STZ groups receive intranasal 1µl/gr saline 0.65% daily. The STZ-IN group received 4 U/40 µl regular insulin daily for 18 days (total of 18 doses), and the STZ-GFRS group received 1µl/gr GFRS every other day for 18 days (total of 9 doses). STZ- IN + GFRS receive 4 U/40 µl regular insulin daily and 1µl/gr GFRS every other day for 18 days. Morris water maze (MWM) tests were done from days 18 to 22 and the open field (OF) trial was done on day 23. Finally, the rats were decapitated after the OF trial, and the brains were rapidly dissected (Fig. 1).

2.3 Intracerebroventricular cannulation

On the zero day of the experiment, intracerebroventricular cannulation of both lateral ventricles was operated. Briefly, anesthesia was induced by an intraperitoneal injection of ketamine HCl 10% (80 mg/kg) and xylazine HCl 2% (10 mg/kg). The head was positioned in a stereotaxic frame and a sagittal incision from the midline of the scalp was made. Then after removing the remaining tissue, bregma, and sagittal line boundaries were distinguished. Holes were drilled in the skull on both lateral ventricles from the Paxinos atlas coordinates (0.8 mm posterior to bregma, 1.5 mm lateral to sagittal line). External cannulas (height of 3 mm) were adjusted to place in the corpus callosum with no disruption of the lateral ventricle. External cannulas were inserted in drilled holes. Then holes and cannulas were covered with dental cement for fixation of the cannula position. The internal cannula (3.6 mm) was connected to the 10 µl Hamilton syringe (Bonaduz, Switzerland) by a short piece of narrow polyethylene tube and then inserted on the tip of the external cannula.

2.4 Preparation of growth factor-rich serum (GFRS)

There is about 96% homology in growth factor proteins between human and rat blood (Upton et al. 1998). Therefore, in this study, human blood was used for the preparation of GFRS. Blood from five healthy young male donors (45 ml each), after informed consent, was obtained through a median cubital vein puncture.

Every 14 ml of venous blood was collected in a tube containing 0.7 ml of 3.8% sodium citrate and then centrifuged at 360 g for 10 min. Separated plasma was collected till just above the buffy coat to prevent mixed-up white blood cells with plasma. Whole blood and the obtained plasma fraction (platelet-rich plasma, PRP) were analyzed with the hematologic analyzer. To activate the platelet for release of its biological factor, 50 µl calcium chloride 10% is added to 1 ml of plasma. Then for completion of the clotting and retracting process, plasma was incubated for 20 min at 37 ˚C and centrifuged at 1000 g for 20 min at 4 ˚C. Obtained supernatant which is known as growth factor-rich serum (GFRS) was aliquoted and stored at -80˚C, until use (Anitua et al. 2016).

2.5 Hematologic parameters

Blood cells were counted with a hematologic analyzer (XP-300, Sysmex, Kobe, Japan) in whole blood and PRP samples from all donors, and reports are summarized in Table 1. Whole blood and PRP samples volumes were measured by the graduated centric tube. GFRS protein concentration was measured by the Bradford method. Insulin-like growth factor 1 (IGF-1) concentration in GFRS was estimated by commercially available colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D). The platelet yield (PY) was calculated from the following equation:

PY = (Platelet number in plasma * Volume of plasma) / (Platelet number in blood * Volume of blood) * 100

Table 1

Hematologic analysis of blood, PRP, and GFRS samples
Blood sample id	1	2	3	4	5	GFRS Pool
Blood platelets *	193	197	241	278	203	-
PRP platelets *	358	344	417	515	349	-
Blood leukocyte *	5.7	5.9	6.8	7.1	6.2	-
PRP leukocytes *	0.5	0.2	0.4	0.6	0.2	-
Platelet yield %	69	65	64	68	63	-
GFRS total protein^#	4.37	4.29	5.26	5.93	4.68	4.82
GFRS igf-1^@	149	125	163	216	132	156

2.6 Intranasal delivery of drugs

Intranasal treatment started on day 4 of the experiment. Intranasal administration was carried out without anesthesia while the rat head was manually extended in a supine position (Marks et al. 2009). The fluid was delivered intranasally by a 10 µl Eppendorf pipette. To ensure fluid inhalation into nares, the rats were held in the same position for a further one minute between drug administrations to each naris.

2.7 Spatial learning and memory evaluation with MWM

MWM trials were done from 18 to the 22nd days of the experiment. The maze is a circular pool (160 cm in diameter, 60 cm in height), and filled with 22 ± 1°C water until 35 cm in height. The pool is divided quarterly into 4 equal arenas to north, south, east, and west, and a point of leaving the animal in the water was marked in each quarter of the pool. A circular transparent platform with a 10 cm diameter, was placed in the middle of the target quadrant 1.0 cm under the pool water height. Rats were trained for platform finding with four trials per day from the 18th to the 21st days of the experiment. Rats entered the maze facing the pool wall. The first trial quadrant was changed each day. The rats were allowed to swim for the hidden platform finding within 60 s, if failed it were directed to the platform. Let the rats stay on the platform for 30 s before exiting from the pool then rest for one minute in the cage. For spatial memory evaluation, a probe trial was accomplished 24 h after the last training day. The hidden platform was removed in this trial and rats were left from the opposite of the target quadrant into the maze for exploratory swim analysis in the 60s. A visible platform trial was carried out after the probe trial to check the rat's vision and platform perception. Latency to escape on the platform in training days, time spent in the target quadrant in the probe trial, and swimming speeds were automatically measured with a video tracking system (EthoVision XT, Noldus Information Technology).

2.8 Anxiety examination with open field trial

On the 23rd day of the experiment, an open field (OF) trial was done for anxiety and exploratory activities evaluation from rats’ exploration of the arena in 15 min. The field was a 75×75 cm dark fiberglass square arena, with 40 cm walls. Each rat was monitored individually with the infrared camera. The time spent in the center area, exploration distance in the arena, and body elongation posture were automatically measured by a video tracking system (EthoVision XT, Noldus Information Technology). The frequency and duration of roaring, grooming, defecation, and urination were evaluated manually (Prut and Belzung 2003). Hyperactivity could be detected in the open-field trial by exploring distance, rearing, and grooming behaviors. Rearing is erect postures, which are related to the exploring intention in rodents. Risk-taking behavior was evaluated by rat exploration time in the center of the field. Risk-assessment behavior was evaluated by measuring stretch body elongation posture frequency in the OF trial. Hyperactivity increased risk-assessment behavior, and decreased risk-taking behavior have been related to anxiety (Conn 2017).

2.9 Hippocampus homogenate preparation

The left hippocampus was removed, weighed, and was homogenized in the 10-fold volume of ice-cold phosphate buffer saline properly; using Homogenizer (IKA T10 basic, Germany) apparatus, samples were homogenized for about 3 minutes, then centrifuged (12000 rpm) at 4°C for 5 min and the supernatant was isolated and stored at -80°C for the following measurement of hippocampus antioxidant enzymes activity, MDA level, and nitrogen metabolites.

2.9.1 Activity estimation of superoxide dismutase (SOD)

SOD activity was estimated by the colorimetric method at 520 nm according to the instructions of the SOD Assay Kit (Zell Bio GmbH, Ulm, Germany).

2.9.2 Activity estimation of glutathione peroxidase (GPx)

GPx activity was assayed by colorimetric method at 340 nm based on the instructions of the GPx Assay kit (ZellBio GmbH, Ulm, Germany).

2.9.3 Estimation of MDA level

Lipid peroxidation in animal tissues was assayed by the thiobarbituric acid reaction method using 1, 1, 3, 3-tetra ethoxy propane as the standard. The pink color formed from the reaction of TBA with MDA. Absorbance was read at 532 nm (Ohkawa et al. 1979).

2.9.4 Nitrogen metabolites assay

This assay relies on a diazotization reaction that was previously described (Green et al. 1982). In this method, first nitrite diazotized with sulfanilamide, at low pH to form diazonium salt. This salt is then reacted with N-naphthyl ethylenediamine, to form a stable compound. The absorbance of this composition at 540 nm is directly proportional to the nitrogen metabolites of the sample.

2.9.5 Protein concentration estimation

For estimation of the total protein concentration of tissue supernatant and GFRS, the bovine serum albumin was utilized as standard in the Bradford method (Kruger 2009). All data were expressed as x/mg protein of tissue.

2.10 Tissue processing protocols

After getting out of the brain, the right hemispheres were fixed with paraformaldehyde 4% buffer, and dehydrated with a solution containing 30% sucrose. Afterward immersed in very cold methyl butane for a few seconds and embedded in OCT in the vapor phase of liquid nitrogen. Then, the hemisphere was sectioned throughout the hippocampus with a thickness of 50 µm coronally, using a cryostat (SLEE, Frankfurt, Germany). Every 7th section (350 µm interval) was selected with systematic random sampling then mounted on gelatin-coated slides and stained with cresyl violet.

2.11 Image acquisition and cell detection

For image acquisition, a 16-megapixel image sensor (SonyExmor RS IMX240) was attached to a Nikon Eclipse 200 microscope. Image data was transported via USB3 port to the computer. Then live image data was imported to Image J software (Fiji) via a webcam capture plugin (Schindelin et al. 2012). Cell type was distinguished by the difference in size, shape, chromatin distribution, and Nissl bodies (Fig. 2). We used a study of the Nissl staining difference in cell types of the rat brain as a reference (García-Cabezas et al. 2016). Pyknosis (nucleus wrinkles and chromatin condensation) and karyorrhexis (dense chromatin fragmentation) are caused by apoptosis and necrosis (Majno and Joris 1995). Neurons with these characteristics were distinguished as dead neurons (Fig. 2)

2.12 Volume estimation

Right hippocampus volume was estimated by the point counting technique based on Cavalieri‘s principle. For that purpose, with the use of the Image J grid plugin, a point-grid system was superimposed onto a live image of each section, and the number of points falling within the region of interest was counted manually. Finally, an unbiased estimate of the volume was calculated from the following equation:

V = T × (a/p) × (∑P)

where ∑P is total point counts, a/p is the area associated with each point, and T is section interval.

The sampling grid size (500 µm × 500 µm) was optimized to achieve a mean Gundersen coefficient of error (CE) of < 10% (Gundersen et al. 1999) (Fig. 3A). In total, 455 ± 63 grid point was counted in the 12–14 section.

2.13 Cell density and total number estimation

Right hippocampus cell density estimated by optical disector method. To apply this method live image was calibrated with a Zeiss micrometer at high power magnification. Then an accurate unbiased brick (50 µm × 50 µm) was randomly superimposed on the live image with the Image J disector plugin (Fig. 3B). The microscope equipped with a Heidenhain electronic microcator with a digital readout for measurements in the z-direction (with a precision of 0.5 µm). Cells were counted in the z-axis with Nikon Plan Apo 100x/1.40 Oil objective. A guard volume on both sides of the section was used to avoid sectioning artifacts. Approximately 200 systematic random locations were counted across all sections for each case to achieve a coefficient of error of less than 10% for all groups. After counting, numerical density is calculated based on the following formula (Howard and Reed 2004).

Nv = ∑Q / [∑P × a(f) × h]

Where Nv is numerical density, ∑Q is total counted cells, ∑P is total counted disectors, a(f) is the area of disector, and h is disector height.

The total number of cells was estimated by multiplying of numerical density (Nv) by the volume of the right hippocampus (V).

2.14 Statistical analysis

Data were expressed as the means ± SEM, and P < 0.05 was considered significant. The normality of data was evaluated by the Kolmogorov-Smirnov test. For assessment of MWM escape latency changes through the learning days, a two-way repeated-measures analysis of variance (TWRM-ANOVA) and for other parameters one-way ANOVA followed by Tukey's post hoc test was utilized for comparisons among various groups. All statistical analyses were performed using GraphPad software (Prism Software Inc., San Diego, CA, USA).

3.1 Morris Water Maze

There was a significant difference in escape latency between STZ- GFRS and the control group on the first day of the acquisition trial (p < 0.05). On the 2nd day, there was a significant difference in escape latency between the control group and other groups (p < 0.0001). On the 3rd day, a significant difference in escape latency between the control and STZ (p < 0.0001), and STZ- GFRS (p < 0.01) groups was observed. Also, on this day escape latency of the STZ- IN group significantly decreased in comparison with the STZ group (p < 0.05). On the last day of acquisition, there was a significant difference in escape latency between the control and other groups except for STZ- IN + GFRS. Our results demonstrated that escape latency in nasal-treated groups was significantly lower than in the STZ group (STZ- IN, p < 0.01; STZ- GFRS, p < 0.05; STZ- IN + GFRS, p < 0.001; 4A). No significant differences were observed in swim speed between groups on all days of the trial (4C). In the probe trial, the percent of the time spent in the target quadrant significantly decreased in STZ, STZ- IN, and STZ- GFRS groups (p < 0.0001, p < 0.05, p < 0.05 respectively) as compared with the control group (4B). Nasal treatments significantly increased the percentage of time in the target quadrant as compared with the STZ group (p < 0.05). Escape latency in visible platform trials was not significantly different between the groups (4D).

3.2 Open field trial

In the 15-minute open field trial, exploration distance in the STZ group was significantly higher than in the control group (p < 0.05). This parameter in the nasal-treated groups significantly decreased in comparison with the STZ group (STZ- IN, p < 0.001; STZ- GFRS, p < 0.01; STZ- IN + GFRS, p < 0.05; Fig. 5A). The STZ group has a significantly lower center and grooming duration in comparison with the control group (p < 0.01). The center and grooming duration of nasal-treated groups significantly increased in comparison with the STZ group (p < 0.05, Fig. 5B, C). Also, center duration increased in STZ- IN + GFRS as compared to STZ- GFRS (p < 0.05). Roaring on the wall, stretch body elongation, and defecation-urination frequency in the STZ group significantly increased as compared with the control group (p < 0.01). The number of rearing on the wall in the nasal-treated groups significantly decreased in comparison with the STZ group (p < 0.05, Fig. 5D). STZ- IN and STZ- GFRS groups have significantly lower stretch body elongation in comparison with the STZ groups (p < 0.05, Fig. 5E). Defecation and urination frequency through the trial decreased in the nasal-treated groups as compared with the STZ group (p < 0.05, Fig. 5F).

3.3 Hippocampus biochemical assessment

MDA concentration (index of lipid peroxidation) in the STZ group was significantly higher than the control group (p < 0.01). In comparison with the STZ group, lipid peroxidation decreased in treated groups but only in the STZ- IN + GFRS group, this reduction was significant (p < 0.05, Fig. 6A).

Nitrogen metabolite concentration significantly increased in STZ (p < 0.05) and STZ- GFRS (p < 0.01) groups as compared with the control group. Co-treatment significantly decreased nitrogen metabolites in the hippocampus as compared with the STZ group (p < 0.05, Fig. 6B).

As shown in Fig. 6C, the hippocampus antioxidant enzyme activity of SOD in the STZ group was significantly lower than control (p < 0.05). Nasal treatment with insulin and GFRS elevates SOD activity in the hippocampus but this increase was not significant. There were no significant differences in GPx activity of the hippocampus among the groups (Fig. 6D).

3.4 Hippocampus histological examination

Histopathological changes in neurons of CA1, CA3, and DG were investigated on cresyl violet stained sections (Fig. 7). The Sample belonging to the Control shows intact pyramidal cells of CA regions with cone-shaped soma and distinct nucleolus, also normal granular cells in DG was observed. Darkly stained neurons with shrunken or sickle-shaped soma and without distinct nucleolus were known as degenerated or dead neurons. Icv-STZ injection caused ventricular enlargement and hippocampal atrophy, neuronal loss, and gliosis, especially in the CA1 region. Also, numerous dead neurons were observed in the subregions indicating neurodegeneration. Nasal treatments visibly improved ventricular enlargement and decreased neuronal loss in the icv-STZ rat's hippocampus. Also, no gliosis was seen but some dead neurons were observed in the hippocampus. Further, co-treatment displayed a greater number of healthy neurons and fewer dead neurons indicating stronger protection against STZ-induced neurotoxicity.

3.5 Hippocampus volume estimation

Results from volume estimation showed that icv-STZ injection decreased the right hippocampus volume but this reduction only in the STZ group became significant in comparison with the control (p < 0.0001); therefore, nasal-treated groups had significantly higher right hippocampus volume in comparison with STZ groups (STZ- IN p < 0.01, STZ- GFRS p < 0.05, STZ- IN + GFRS p < 0.01) (Fig. 8).

3.6 Hippocampus cell number estimation

3.6.1 Neuron number

As shown in Fig. 9A, the STZ group had lower neurons in the right hippocampus in comparison with the control group (p < 0.0001). All nasal-treated groups had significantly more right hippocampus neurons than the STZ group (p < 0.01).

3.6.2 Dead neuron number

The total dead neuron number of the right hippocampus in the STZ group was significantly higher than the control (p < 0.0001). Nasal-treated groups had significantly lower dead neuron numbers in the right hippocampus in comparison to the STZ group (p < 0.05) (Fig. 9B). There were fewer dead neurons in the co-treatment groups than in the STZ-GFRS group (p < 0.05).

3.6.3 Oligodendrocyte number

The STZ group had lower oligodendrocytes in the right hippocampus as compared with the control group (p < 0.0001). All nasal-treated groups had significantly greater right hippocampus oligodendrocytes than the STZ group (STZ- IN p < 0.05, STZ- GFRS p < 0.001, STZ- IN + GFRS p < 0.0001) (Fig. 9C).

3.6.4 Astrocyte number

The right hippocampus astrocyte number in the STZ group was significantly higher than the control (p < 0.001). Nasal insulin-treated groups had significantly lower astrocyte numbers in the right hippocampus in comparison to the STZ group (STZ- IN p < 0.01, STZ- IN + GFRS p < 0.05) (Fig. 9D).

3.6.5 Microglia number

The total microglia number of the right hippocampus was estimated in different groups (Fig. 9E). Microglia number in the right hippocampus of the STZ group was significantly higher than the control (p < 0.0001). Treated groups had significantly lower microglia numbers in the right hippocampus in comparison to the STZ group (p < 0.05).

3.6.6 Endothelial cell number

As shown in Fig. 9F, the GFRS-treated groups had a significantly higher endothelial cell number in the right hippocampus in comparison to the control (p < 0.05).

The present study was carried out to investigate the therapeutic effects of intra-nasal GFRS or insulin and their co-treatment in icv-STZ-induced memory and behavior impairments in rats and explore the correlation of hippocampus volume, cells number, and oxidative-nitrosative stress with these behavioral changes.

The MWM is a reward conditioning related to spatial memory performance and is considered an accepted model for the examination of learning and memory in rodents (Morris 1984). The STZ group escape latency was not changed during the training days (Fig. 4A) and the time percent in the target quadrant was lower than the control group in the probe trial (Fig. 4B). These signs are related to learning/memory impairment and agree with a previous study (Awasthi et al. 2010; C Correia et al. 2013). In the present study, after intranasal treatment of insulin, learning/memory function improved, as proven by escape latency reduction during training days (Fig. 4A) and time percent in the target quadrant elevation during the probe trial (Fig. 4B). A study reported that insulin signaling modulates learning and memory (Zhao et al. 2004). In line with our findings, another study has shown that intranasal administration of insulin in rats improves spatial memory defects induced by icv-STZ injection (Rajasekar et al. 2017). Platelets contain growth factors and several neurotransmitters that are necessary for brain cell communication, like dopamine, histamine, serotonin, epinephrine, g-aminobutyric acid (GABA), and glutamate (Hayon et al. 2013). Neural and glial cells have receptors for these substances and nasal absorption of them previously described. Therefore, nasal GFRS could have beneficial effects for the treatment of defects induced by icv-STZ. We could not find any study that has used intranasal GFRS for the treatment of the icv-STZ rat model. However, many studies showed the neuroprotective effect of growth factors in various brain damage models. For example, it was reported that VEGF through angiogenesis induction improves memory impairment in APP/PS1 mice (Wang et al. 2011). Moreover, it was demonstrated that hepatocyte growth factor (HGF) gene therapy ameliorates cognitive deficits in an animal model of AD (Takeuchi et al. 2008). Previous studies have shown that IGF-1 enhances Ras signaling through PI3K activation and facilitates LTP in a Ras-ERK–dependent manner hence improving synaptic plasticity and spatial memory (Desbois-Mouthon et al. 2001; Patterson et al. 2010). A study reported that growth factors derived from platelets improved memory defects in APP/PS1 mice (Anitua et al. 2014). In line with these growth factor studies, our nasal GFRS treatment improved spatial memory, however, its effect was not as much as insulin (Fig. 4A). Co-treatment (insulin + GFRS) had a better memory improvement effect verified by no significant differences in escape latency of the last training day and percent time in the target quadrant as compared to the control group (Fig. 4A, B). As icv-STZ suppresses insulin signaling in the brain cells, so, targeting different signaling pathways with co-treatment of insulin and GFRS has better improvement in spatial memory.

Behavior impairments have been reported in animals that received icv-STZ (Suzanne 2009). Also, a study reported anxiety behavior in the icv-STZ rats (Gutierres et al. 2014). The open field trial was originally described by Hall et al for the emotional examination of rats (Hall 1936). Spending more time in the center of the arena is linked with anxiolysis (Walsh and Cummins 1976). In the present study, icv-STZ injection induces anxiogenic behavior in rats as proven by hyperactivity, decreased center duration and grooming duration, and increased roaring and body elongation frequency (Fig. 5). Because the density of insulin receptors in the cortical and subventricular regions of the limbic system is high, it is expected that insulin will affect mood, reward, motivation, and perception (Nguyen et al. 2018). We found the anxiolysis effect of nasal insulin treatment, like reduced activity and increased center duration (Fig. 5). This effect was not previously studied in the icv-STZ model to compare but in agreement with our results, a study reported that prolonged fasting raises brain insulin levels and causes anxiety reduction to focus on the exploration of novel environments (Fadool et al. 2000). Marks et al also observed anxiolysis after awake intranasal administration of insulin (Marks et al. 2009). A correlation between anxiety and the reduction of brain IGF-1 levels is found in STZ-induced diabetes (Aksu et al. 2012). It was shown that the icv administration of IGF-1 improved neurobehavioral defects induced by hypoxia (Lin et al. 2005). It has been demonstrated that the hippocampus regulates the hypothalamic-pituitary-adrenal axis (HPA) through FGF2 signaling (Salmaso et al. 2016). BNDF and VEGF signaling are concerned with lessening anxiety behavior in rodents (Uysal et al. 2011). Consistent with this evidence, the present study revealed that nasal GFRS improves anxiogenic behavior. GFRS contains other factors like serotonin and GABA which could induce anxiolysis (Leiter and Walker 2020). The anxiolytic effect of the co-treatment was better than other treated groups as evidenced by a significant center duration increase (Fig. 5B). Deng et al (2009) demonstrated that icv-STZ injection reduces the sensitivity to insulin in the rat brain (Deng et al. 2009). Co-treatment could activate an insulin-related signaling pathway in different ways so may have better anxiolysis.

Free radical production and oxidative stress are considered important factors for memory and behavior defects induced by icv-STZ in rats (Gutierres et al. 2014). A previous study reported a nitrite level elevation in icv-STZ rats’ brains (Tiwari et al. 2009). Biochemical investigations demonstrated a significant MDA elevation and activity reduction of antioxidant enzymes (e.g. SOD, GPx) in icv-STZ rats’ brains and revealed neuronal damage produced by oxidative-nitrosative stress (Mansouri et al. 2013) that are in line with our findings which icv-STZ injection elevates lipid peroxidation as well as NO production and reduces SOD activity in the hippocampus (Fig. 6). In brain cells, insulin regulates the inflammatory response, and cytokine secretion and increases glucose harvesting from the blood-brain barrier (Bedse et al. 2015a). In the present study, nasal treatment of insulin improved oxidative stress in the hippocampus of icv-STZ injected rats but no significant difference was detected (Fig. 6A). Which may be a result of some insulin resistance induced with STZ and nasal treatment. It is demonstrated that STZ suppresses the tyrosine kinase activity of insulin receptors by releasing NO intracellularly, which leads to S-nitrosylation of receptor tyrosine kinase (Simon et al. 1996) but insulin strengthens nitric oxide-dependent vasodilation and increases cerebral blood flow. Previous studies reported the protective effects of PRP growth factors on oxidative damage to bone, ovary, and muscle tissues (Bakacak et al. 2016; Martins et al. 2016; Tohidnezhad et al. 2014). Several growth factors released from platelets (HGF, IGF-1) can trigger the PI3K/Akt signaling pathway which leads to ROS reduction and oxidation resistance increase in cells (Kajstura et al. 2001; Zhou et al. 2009). A study showed that PDGF had neuroprotective effects against oxidative stress through insulin signaling pathway activation (Zheng et al. 2010). In the present study, some hippocampus oxidative stress improvements in nasal GFRS-treated rats were observed, however, insulin had a better-improving effect on nitrosative stress of the hippocampus than GFRS (Fig. 6B). Although nasal treatment of insulin or GFRS improved oxidative stress, only co-treatment significantly decreased oxidative-nitrosative stress in the hippocampus of icv-STZ injected rats (Fig. 6A, B). We could not find any study that has used a co-treatment of insulin and GFRS to compare but it was reported that insulin and IGF-1 reduce oxidative stress and neuroinflammation in the hippocampus of Alzheimer's models (Rivera et al. 2005). IGF-1 had a regulatory role in the FoxO signaling pathway (O’Neill et al. 2016). Transcription factors of this pathway are vital regulators of cellular response to oxidative stress and pathway changes was seen in insulin resistance and AD (Manolopoulos et al. 2010).

Human AD studies showed that hippocampus volume reduction was correlated with global cognitive function and memory performance impairments (Elgh et al. 2006; Su et al. 2018). It was reported that the icv-STZ injection in rats, significantly reduced the Stratum radiatum volume of the dorsal hippocampus and severely decreased neurons cell body volume and hippocampal CA1 pyramidal layer. In fact, these remarkable changes are signs of hippocampus atrophy and shrinkage (Zappa Villar et al. 2018). In agreement with the mentioned studies, we observed a significant reduction of hippocampus volume and tissue atrophy and shrinkage in the STZ group (Fig. 8). We could not find any study for nasal insulin and GFRS effects on hippocampus volume but human studies report that hippocampus volume decreased in peripheral insulin resistance patients (Bruehl et al. 2009) and human serum IGF-1 levels correlated to brain atrophy and dementia (Westwood et al. 2014). In agreement, nasal insulin and GFRS prevented hippocampus atrophy and volume reduction in icv-STZ-injected rats (Fig. 8).

One of the characteristic hallmarks of Alzheimer's disease is inflammation and neuronal loss, especially in the hippocampus. The inflammatory process in AD patients manifests in the morphological changes in microglia and astrocytes or an increase in their number, size, and motility(Meraz-Ríos et al. 2013). In animal models of sAD an increase in the number of apoptotic cells was detected in the hippocampus of icv-STZ-injected rats (Hashemi-Firouzi et al. 2017). It was demonstrated that icv-STZ injection induces neurotoxicity through damage to myelin and oligodendrocyte (Shoham et al. 2003). Other studies showed microglia, astrocyte, and dead cell number elevation as well as neurogenesis reduction in the hippocampus of the icv-STZ injected rats which was caused by induction of neuroinflammation and insulin signaling disorder (Bassani et al. 2018; Mishra et al. 2018; Rostami et al. 2017). In agreement with the mentioned studies, our results showed that the survival rate of neurons and oligodendrocytes decreased in STZ groups as evidenced by neuron and oligodendrocyte number reduction and dead neuron elevation in the hippocampus of icv-STZ rats (Fig. 9A, B, C). Also, we found an increase in microglia and astrocyte number in the hippocampus of icv-STZ rats that may result from STZ-induced neuroinflammation (Fig. 9D, E).

A study reported that icv-STZ injection severely impairs growth factor receptor expression in the frontal cortex and hippocampus; also concluded this damage may reason for the loss of oligodendrocytes and neurons in the icv-STZ model. It was shown that intranasal insulin significantly ameliorated neuron loss and gliosis in the hippocampal CA1 and CA3 regions of icv -STZ rats (Chen et al. 2018). Previous studies reported that insulin stimulates glucose metabolism in the brain, regulates neuronal and oligodendroglia cell survival, also modulates neuroinflammation (De la Monte 2017). In the present study, intranasal insulin increased the survival rate of the neuron and oligodendrocyte (Fig. 9A, B, C) and also decreased astrocyte and microglia numbers in the hippocampus of icv-STZ rats (Fig. 9D, E). The anti-inflammatory effect of intranasal insulin seems to be better than GFRS as evidenced by significant lower microglia number in the hippocampus.

Several growth factors present in GFRS including NGF, FGF, BDNF, PDGF, VEGF, IGF-1, and TGF-b alone or in combination have been shown to induce neuroprotective and antiapoptotic effects (Emel et al. 2011; Luo et al. 2012; Won et al. 2006). Previous studies described that IGF-1 plays an important role in cell survival and can inhibit the pro-apoptotic and inflammatory agents in ischemic brains through the PI3K/Akt pathway (Fukunaga and Kawano 2003). It was demonstrated that intranasal IGF-1 could improve oligodendroglia survival (Cai et al. 2011). A study showed that PDGF induces neuroprotection, and also improves angiogenesis and vascular remodeling after cerebral ischemia (Krupinski et al. 1997). In line with this evidence, intranasal administration of GFRS, significantly increased neuron and oligodendrocyte numbers and decreased microglia and dead neuron number in the hippocampus of icv-STZ rats (Fig. 9A, B, C). GFRS effect on oligodendrocyte survival was better than insulin as evidenced by STZ- IN remaining significant with the control while GFRS-treated groups did not (Fig. 9C). Also, GFRS-treated groups had significantly higher endothelial cell numbers as compared to the control (Fig. 9F). In the present study, we found that the improving effect of insulin and GFRS co-treatment on neuronal survival is stronger than single therapy which is evidenced by no significant difference between STZ- IN + GFRS and the control groups (Fig. 9A), and also a significant difference in dead neuron number observed between GFRS single and co-treated groups (Fig. 9B). Studies reported that exogenous administration of TGF-β1 elevates the potency of other neurotrophic factors through modulation of their action and signaling (Sometani et al. 2001; Unsicker and Krieglstein 2000). Considering icv-STZ could disrupt both insulin and other growth factors related signaling in the brain cells, insulin receptors are saturable, and after a long treatment, cells become insulin resistant which may worsen the signaling disorder induced by STZ, so, maybe co-treatment had more improvement because both insulin and GFRS could affect different signaling pathways like PI3K-AKT, ERK, FoxO, GSK3B, MAPK, and CREB.

Study limitations

Previous studies about insulin and growth factor treatment utilize real-time PCR, western blot, and immunohistochemistry techniques to investigate treatment effects from gene expression to cellular receptor density but unfortunately, due to budget limitations cellular signaling changes were not studied.

Despite of mentioned limitations, from the obtained results, it could be concluded that the administration of intranasal insulin or GFRS prevents STZ-induced memory impairment and improves anxiogenic behavior. However, co-treatment, somehow, has a stronger effect, especially on neuronal survival and oxidative stress reduction in comparison with a single therapy. The improving effect of intranasal insulin and GFRS on spatial memory and anxiety could be attributed to the reduction of oxidative stress and neuroinflammation in the hippocampus.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All experimental procedures in the study were based on the National Institutes of Health’s Guide for Care and Use of Laboratory Animals and the Animal Research: Reporting in Vivo Experiments (ARRIVE) Guidelines and approved by the local Ethical Committee of the Shiraz University of Medical Sciences (approval number: IR.SUMS.REC.1398.168).

CONSENT FOR PUBLICATION

Not applicable

AVAILABILITY OF DATA AND MATERIALS

Study data and analysis are available from the corresponding author upon reasonable request.

COMPETING INTERESTS

The authors declare that they do not have any known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

FUNDING

This research was financed by grant number SUMS-97-01-01018475, awarded to Dr. Ali Rafati from the Shiraz University of Medical Sciences, Shiraz, Iran.

AUTHORS' CONTRIBUTIONS

Mahdi Khorsand Ghaffari: designing the study, performing laboratory work, data collection, and writing the manuscript draft. Ali Rafati: supervising laboratory works, conceptualization, methodology, and data analysis. Narges Karbalaei: conceptualization, methodology. Masoud Haghani: conceptualization, methodology. Marzieh Nemati: methodology, laboratory assistance, data analysis. Niloofar Sefati: methodology, laboratory assistance, graphical design. Mohammad Reza Namavar: conceptualization, methodology, data analysis, editing, and revising the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGEMENTS

The present article is part of a Master of Sciences thesis written by Mahdi Khorsand Ghaffari.

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The effect of intra-nasal co-treatment with insulin and growth factor-rich serum on behavioral defects, hippocampus histological, and oxidative-nitrosative stress changes induced by icv-STZ in a rat model

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Abstract

Figures

1 Introduction

2 Material & Method

2.1 Animals

2.2 Experimental design

2.3 Intracerebroventricular cannulation

2.4 Preparation of growth factor-rich serum (GFRS)

2.5 Hematologic parameters

2.6 Intranasal delivery of drugs

2.7 Spatial learning and memory evaluation with MWM

2.8 Anxiety examination with open field trial

2.9 Hippocampus homogenate preparation

2.9.1 Activity estimation of superoxide dismutase (SOD)

2.9.2 Activity estimation of glutathione peroxidase (GPx)

2.9.3 Estimation of MDA level

2.9.4 Nitrogen metabolites assay

2.9.5 Protein concentration estimation

2.10 Tissue processing protocols

2.11 Image acquisition and cell detection

2.12 Volume estimation

2.13 Cell density and total number estimation

2.14 Statistical analysis

3 Results

3.1 Morris Water Maze

3.2 Open field trial

3.3 Hippocampus biochemical assessment

3.4 Hippocampus histological examination

3.5 Hippocampus volume estimation

3.6 Hippocampus cell number estimation

3.6.1 Neuron number

3.6.2 Dead neuron number

3.6.3 Oligodendrocyte number

3.6.4 Astrocyte number

3.6.5 Microglia number

3.6.6 Endothelial cell number

4 Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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