{"id":62120,"date":"2024-12-30T11:42:16","date_gmt":"2024-12-30T11:42:16","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=62120"},"modified":"2025-01-06T18:11:11","modified_gmt":"2025-01-06T18:11:11","slug":"nasal-treatment-of-standardized-centella-asiatica-leaves-extract-ameliorates-chronic-unpredictable-mild-stress-in-laboratory-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no4\/nasal-treatment-of-standardized-centella-asiatica-leaves-extract-ameliorates-chronic-unpredictable-mild-stress-in-laboratory-rats\/","title":{"rendered":"Nasal Treatment of Standardized Centella Asiatica Leaves Extract Ameliorates Chronic Unpredictable Mild Stress in Laboratory Rats"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Individuals often encounter stressors in\ntheir daily lives, which could affect mood, focus, and performance. However,\nchronic exposure can lead to more serious consequences, including cognitive and\nbehavioral changes, such as cognitive decline1<\/sup>.\nChronic stress disrupts the body’s ability to maintain stability (allostasis)\nand activates many physiological changes, such as stimulation of the\nsympathetic nervous system and inflammation, ultimately compromising\ncardiovascular and immune functions2, 3<\/sup>.\nThese physiological consequences significantly reduce quality of life,\nhighlighting the need for interventions that target stress-induced\nphysiological dysregulation to improve overall well-being4<\/sup>.<\/p>\n\n\n\n

The symptoms of stress-induced behavioral\nand cognitive decline follow distinct temporal profiles. Within minutes to\nhours of stress induction, anxiety emerges as the initial response and is\ncharacterized by heightened worry, nervousness, and physical tension5<\/sup>. Activation of “hypothalamic-pituitary-adrenal” (HPA)\naxis promotes stress hormones, such as cortisol6<\/sup>. In the short term (hours to days), working memory becomes\ncompromised, impacting the ability to conduct routine tasks, especially those\nthat require manipulation information in the mind. This is thought to be due to\nstress-induced disruptions in the prefrontal cortex, a key region in working\nmemory function7<\/sup>.<\/p>\n\n\n\n

The intermediate stages (days to weeks)\nshowed a decline in spatial memory and ability to navigate and remember\nlocations. Chronic stress can disrupt the hippocampus, a critical brain\nstructure for spatial navigation, potentially through mechanisms involving\nreduced neurogenesis (neurogenesis refers to the birth of new neurons)8<\/sup>. While long-term memory initially appears to be less affected,\nchronic stress can impair the consolidation of new information into long-term\nstorage over weeks or months9<\/sup>. This extended stress exposure may further contribute to the\ndevelopment of depression, a more prolonged state characterized by low mood,\nanhedonia (loss of pleasure), and sleep and appetite disturbances10<\/sup> . Chronic stress alters neurotransmitter systems, such as serotonin\nand norepinephrine, to pose a risk of depression development11<\/sup>. Finally, although less prevalent, chronic stress can also lead to\nincreased aggression or irritability, highlighting the diverse behavioral consequences\nof prolonged stress exposure12<\/sup>. <\/p>\n\n\n\n

Chronic stress is known to disrupt the\nHPA axis and the body’s stress response system13<\/sup>. Dysregulation of the HPA axis can have far-reaching effects on\nvarious physiological systems, including emotional regulation, eating habits,\nand the immune system14<\/sup>. HPA axis stimulation secretes glucocorticoids, such as cortisol,\nwhich play a crucial role in redirecting energy resources to cope with stress15<\/sup>. Increased cytokines lead to depression both directly (HPA axis)\nand indirectly (cytokines)16<\/sup>. Cytokines directly act on hypothalamic and pituitary cells and\nincrease the production of CRF, ACTH, and cortisol17<\/sup>. Dysregulation of 5HT transmission in the cortex is also associated\nwith chronic stress18<\/sup>. During chronic stress, microglial activation occurs, which leads\nto increased cytokines, serotonin, and inflammation19<\/sup>. <\/p>\n\n\n\n

The management of stress-related\ndisorders is limited to symptomatic treatment without addressing the underlying\ncause (stress) with undesirable side effects20<\/sup>. Medication options addressing HPA axis dysregulation, a cause and\nimportant target of stress-related disorders, with serotonergic efficacy and an\nexcellent safety profile can address chronic stress, and related behavioral\nsymptoms can offer efficient interventions and enhance patient outcomes21<\/sup>. <\/p>\n\n\n\n

Considerable amount of evidence in last\ndecade demonstrated  the efficacy of Gotu\nkola, Centella asiatica<\/em> (L.) Urban, (i.e. CA) leaves (Family: Apiaceae)\nagainst the symptoms of many stress-induced neuropsychological disorders22, 23<\/sup>\nprobably HPA axis, serotonergic neurotransmission, and regulation of brain\ncortisol levels24<\/sup>. The\nsource of CA leaves from Madagascar region contains the major bioactive\ntriterpenoids, such as namely asiaticoside, and madecassoside that are\nbeneficial for the management of many nervous system disorders22, 25<\/sup>. In\naddition, triterpenoid-based standardized CA leaf extract (INDCA) oral\nsupplementation has been reported to alleviate experimental depression26<\/sup> and migraine27<\/sup> via serotonin agonist action27<\/sup>. <\/p>\n\n\n\n

Recently, nasal route has emerged as a\npromising choice for safer and more effective patient-compliant solutions\nagainst stress-related disorders owing to its many advantages, such as\navoidance of first-pass metabolism28<\/sup>. The olfactory perineural space contains transporters that enhance\ndrug transport into the brain29<\/sup>. The intranasal delivery of various compounds, including natural\nbioactive phytoconstituents such as quercetin, has been reported to deliver\ntherapeutic agents to the brain30<\/sup>.<\/p>\n\n\n\n

INDCA nasal solution (INDCA-NS) showed\nrobust safety at doses of 100 \u00b5g\/rat\/day in rats31<\/sup>. In addition, INDCA-NS has been reported to alleviate pain caused\nby nitroglycerine-induced migraine27<\/sup>.  However, the potential of\nINDCA-NS to provide relief from chronic stress has not been explored. <\/p>\n\n\n\n

Hence, our study aimed to evaluate the efficacy\nof nasal INDCA-NS against \u201cchronic unpredictable mild stress\u201d (CUMS), a type of\nchronic stress with good reliability and clinical validity32<\/sup>. <\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Animals<\/strong><\/p>\n\n\n\n

The study utilized Sprague-Dawley rats\n(both genders, 200-250 g weights). Rats were purchased from Crystal Biological\nSolutions (Pune, India). This study was conducted in accordance with Indian\nguidelines on animal ethics33<\/sup>. The\nrats were housed in polypropylene cages in groups of six and maintained in a\ncontrolled environment, as recommended34<\/sup>. The rats were provided with unrestricted access to drinking water\nand feed pellets supplied by Nutrivet Life Sciences (Pune, India). The\nexperimental protocols were sanctioned by the \u201cInstitutional Animal Ethics Committee\u201d\n(IAEC) of \u201cPoona College of Pharmacy, Bharati Vidyapeeth Deemed University,\u201d\nPune, India (approval number CPCSEA\/ PCP\/PCL33\/2018-19). Each rat was used once\nand acclimatized to conditions before the experiment. To eliminate potential\nbias, the observer was blinded to the treatment. All observations were\nconducted between 9:00 AM and 4:00 PM. <\/p>\n\n\n\n

Drugs and Chemicals<\/strong><\/p>\n\n\n\n

Buspirone hydrochloride (Cat No: B7148,\nSigma-Aldrich Chemicals Pvt. Ltd., USA) was used as a positive control. ELISA\nkits for estimating brain-derived neurotrophic factor (BDNF; Catalog No:\nEK0308; Boster Immunoleader, Pleasanton, CA, USA) and cortisol (Catalog number\nCSB-E05112r; Cusabio, USA) were purchased from a local distributor (GK\nBioScience, Pune, India).<\/p>\n\n\n\n

The test solution, INDCA-NS<\/strong><\/p>\n\n\n\n

INDCA-NS was provided by Indus Biotech\nLimited (Pune, India) as a stock solution (1 mg\/ml, 1000 \u00b5g \/1000 \u00b5L)\ncontaining INDCA. Because of intranasal route, INDCA-NS was administered at a\nvolume of 25 \u00b5L\/nostril or 50 \u00b5L per rat as a safe intranasal volume in rats 35<\/sup>. To\nmaintain the same volume per nostril, the stock solution was diluted 10-, 3-,\nand 0-times to deliver final doses of INDCA-NS of 2.5, 7.5, and 10\n\u00b5g\/nostril\/twice daily, that is, 10, 30, and 100 \u00b5g\/rat\/day, respectively.  <\/p>\n\n\n\n

The positive control, BUS<\/strong><\/p>\n\n\n\n

The buspirone hydrochloride solution\n(BUS) was prepared and used for nasal administration as per a previously\nreported procedure36<\/sup>. BUS was prepared by dissolving 15.5 mg of buspirone hydrochloride\nin 5 ml of sterile 0.5% sodium chloride solution and filtering through a\nsterile (0.2 mm) membrane filter for a final concentration of 3.1 mg\/ml. The\nintranasal dose of 20 \u00b5L of BUS, which is equivalent to 0.25 mg\/kg of BUS\n(considering maximum weight of rat = 250 g), was used as a positive control\nbased on past report36<\/sup>.<\/p>\n\n\n\n

Grouping and Treatment Schedule<\/strong><\/p>\n\n\n\n

Adult rats were randomly divided into six\ngroups of 12 rats (G1\u2013G6). G1 (vehicle control, VC) was treated with vehicle\n(25 \u00b5L\/nostril, twice daily, intranasally) for 49 days. Groups G2\u2013G6 were\nsubjected to CUMS for 49 days (D0 to D49), and parameters were measured during\nD35\u2013D49. G2 (stress control, SC) was treated with vehicle (25 \u00b5L\/nostril, twice\na day, intranasal). G3 (BUS group) was served as a positive control and was\ntreated with buspirone (0.25 mg\/rat, twice a day, intranasally). G4 to G6 were\ntreated with INDCA-NS (5,15,50 microgram\/rat\/day, twice a day, intranasal) at\nfinal doses of 10, 30, and 100 microgram\/rat\/day. The CUMS induction, treatment\ndetails, and outcome measures are presented in Figure 1.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Study flowchart for chronic mild unpredictable stress (CUMS) induction, treatments, and outcome measures.<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n

CUMS induction and outcome measures<\/strong><\/p>\n\n\n\n

Training Phase<\/strong><\/p>\n\n\n\n

CUMS was induced in rats as previously reported method37, 38<\/sup>. Briefly, CUMS was induced in the training phase of one week (1% sucrose solution with no food and water for 48 h and then without sucrose solution for an additional 18 h). Thereafter, the rats were housed individually, and sucrose consumption was measured for one hour on each of the following five consecutive days. Food and water were provided ad libitum for five hours. Sucrose consumption (intake) was calculated as the difference in bottle weights (pre- and post-training) and the animal’s body weight (g\/kg) at the end of the training phase. Based on the sucrose consumption test, rats were allotted to non-stressed (G1:VC) and stressed (G2\u2013G6) groups. <\/p>\n\n\n\n

CUMS Induction Phase<\/strong><\/p>\n\n\n\n

After the first week (testing phase), all rats except those in the VC group were subjected to CUMS as a series of unpredictable stresses for a period of seven weeks (49 days) from D0 to D49 of the study, in which treatments were administered for the last 14 days (D36 to D49). After the first week, the sequence of mild and unpredictable stressors was randomly repeated. These stressors included exposure: 10 h – 45\u00b0 tilted cage, 24 h – water \/feed deprivation, 1 h – limited food, 1 h – empty bottles, 24 h – wet cages, and 24 h – continuous light. During the stress sessions, the animals were housed in groups of six per cage, except for sucrose consumption (single housing).<\/p>\n\n\n\n

Measurements<\/strong><\/p>\n\n\n\n

Baseline measurements from behavioral tests, such as the marble burying test (MBT) for anxiety, Y-maze for worming memory, morris water maze (MWM) for special memory, and resident intrusion test (RIT) for aggression, were performed on D0, and sucrose preference tests (SFT) for anhedonia were performed on D5. These behavioral tests were conducted on D35, D42, and D49. On D49, rats were sacrificed, brains were removed, to prepare homogenate with phosphate buffer for cortisol and BDNF estimation.<\/p>\n\n\n\n

Marble Burying Test (MBT) <\/strong><\/p>\n\n\n\n

The effects of treatments on early anxiety were assessed using an earlier reported method39<\/sup>. Briefly, the rats were placed in cages filled with husk bedding material cobs (5 cm height). The test was performed in two phases: habituation and testing. In habituation phase, rats were exposed to cages without marbles. During the testing phase, 20 glass marbles (plain black glass) were evenly placed in the cage on the bedding surface. The rats could explore cage for 20 min and were then removed. The number of marbles in two-thirds deep was counted. <\/p>\n\n\n\n

Y maze<\/strong><\/p>\n\n\n\n

Working memory function was measured using the Y-maze, as described previously 40<\/sup>. The maze consisted of three Y-shaped arms. The three arms were randomly assigned: one was left open, the second was to keep the rat initially, and the third was blocked during the initial training period. During trials, visual cues were added to the maze. Before testing, rats were pre-trained with the novel arm closed. After a 1-hour break, the test proceeded with the novel arm open. The rats’ novelty versus familiarity was evaluated by comparing their behavior in all three arms. The number of visits and time spent in each arm were recorded for 5 min using a camera.<\/p>\n\n\n\n

MWM test<\/strong><\/p>\n\n\n\n

Spatial memory measurement using the MWM test was performed as per an earlier procedure41<\/sup>. MWM, a circular water tank (diameter X height – 150 cm X 45 cm), filled with water (upto 35 cm at  20-22\u00b0C). The tank was divided into four equal sections and had a central round platform (15 cm diameter). Initially, training was given for five trials on five consecutive days, followed by a probe trial (memory retrieval trial) on D6. During the first 60 s, rats freely explored and searched for the hidden fixed platform (2 cm below the water surface), and the time to reach platform was noted as \u201cescape latency.\u201d If a rat was unsuccessful at reaching the platform, an additional 10 s were given before removal. On day 6, a probe trial was performed with opaque water using a nontoxic white color. The rats were permitted to explore the platform, and escape latency and path length were measured using a video tracking system (VJ Instruments, Karanja, India).<\/p>\n\n\n\n

SPT <\/strong><\/p>\n\n\n\n

The SPT was performed using reported procedure37<\/sup>. Before the initiation of the experiments, the SPT was performed with overnight (12 h) fasted rats. At binging and every 7 days of study, the rats were individually placed in cages, where they were exposed to two 250 ml (one with 1% sucrose in distilled water and the second with distilled water). The animals were allowed to drink water for 1 h, with an interchange of bottles after 30 min (to avoid side preference). The percentage of sucrose preference was calculated as follows: [sucrose intake (mL)\/(sucrose intake (mL) + water intake (mL)] \u00d7 100 42-44<\/sup>.<\/p>\n\n\n\n

Resident Intruder Test (RIT)  <\/strong><\/p>\n\n\n\n

As previously reported, RIT was used to evaluate the effects of the treatments on aggression45<\/sup>. This test was carried out in two phases: first, resident rats could explore the cage for 15 m (adaptation), and then, the intruder rat of lesser weight (100\u2013150 g) was transferred to the resident\u2019s cage for 5 m, and attack latency measurements were performed. Rats were considered aggressive when they displayed either a bite or lateral attack.<\/p>\n\n\n\n

Cortisol and BDNF estimation in hypothalamus<\/strong><\/p>\n\n\n\n

After treatment, the rats were sacrificed, and brains were immediately isolated on ice slabs. The hippocampal region was separated, and homogenization was performed using phosphate buffer. The supernatants were divided and used for the estimation of cortisol and BDNF levels using an ELISA plate reader (LisaScan II, Erba Mannheim Diagnostic, London, United Kingdom), ELISA kits, and assay instructions. The results are expressed per gram as pg\/g for BDNF and ng\/g for cortisol.<\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

The data are expressed as mean \u00b1 standard error of means (SEM) using analyzed statistically with Prism 6 (GraphPad; La Jolla, USA) as follows (1) SPT, MWM, RIT, MBT, and OFT: two-way repeated measures analysis of variance (ANOVA) and Bonferroni’s test (2) Cortisol and BDNF: one-way ANOVA and Dunnett\u2019s test. Statistical significance was set at P < 0.05.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Effect on MBT Parameters<\/strong><\/p>\n\n\n\n

ANOVA of MBT data showed significant\neffects of time (F(2.445, 188.3) = 188.7, P < 0.001) and treatment (F(6, 77)\n= 24.52, P < 0.001) on the number of buried marbles (Table 1).\nDunnett’s test revealed no significant difference in the number of buried\nmarbles (between any pair of treatments on D0). The number of buried marbles\nwas significantly higher in the SC group than in the VC group on D35, D42, and\nD49 (P < 0.001). On D35, there was no significant difference between buried\nmarbles in the BUS, INDCA-NS (vs. SC). However, the number of buried marbles\nwas significantly different (P < 0.01 or P < 0.01) in the BUS- and\nINDCA-NS-treated groups compared to SC on D42 and D49. <\/p>\n\n\n\n

Table 1: Effects on early to short-term symptoms of CUMS<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Day<\/strong><\/p>\n<\/td>\n

\n

VC<\/strong><\/p>\n<\/td>\n

\n

SC<\/strong><\/p>\n<\/td>\n

\n

BUS<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(10)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(30)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(100)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Anxiety – Number of marbles buried – MBT<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

3.17 \u00b1
0.78<\/p>\n<\/td>\n

\n

3.50 \u00b1
0.77<\/p>\n<\/td>\n

\n

2.25 \u00b1
0.65<\/p>\n<\/td>\n

\n

1.33 \u00b1
0.40<\/p>\n<\/td>\n

\n

2.67 \u00b1
0.77<\/p>\n<\/td>\n

\n

2.67 \u00b1
0.54<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

1.00 \u00b1
0.39<\/p>\n<\/td>\n

\n

12.17 \u00b1
1.28###<\/sup><\/p>\n<\/td>\n

\n

12.17 \u00b1
1.00<\/p>\n<\/td>\n

\n

13.83 \u00b1
0.80<\/p>\n<\/td>\n

\n

14.25 \u00b1
0.74<\/p>\n<\/td>\n

\n

15.25 \u00b1
1.12<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

1.67 \u00b1
0.47<\/p>\n<\/td>\n

\n

12.08 \u00b1
0.93###<\/sup><\/p>\n<\/td>\n

\n

3.83 \u00b1
0.60***<\/sup><\/p>\n<\/td>\n

\n

6.92 \u00b1
0.72**<\/sup><\/p>\n<\/td>\n

\n

5.08 \u00b1
0.83***<\/sup><\/p>\n<\/td>\n

\n

4.58 \u00b1
0.57***<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

1.83 \u00b1
0.44<\/p>\n<\/td>\n

\n

13.00 \u00b1
1.30###<\/sup><\/p>\n<\/td>\n

\n

2.08 \u00b1
0.40***<\/sup><\/p>\n<\/td>\n

\n

5.08 \u00b1
0.63***<\/sup><\/p>\n<\/td>\n

\n

3.58 \u00b1
0.54***<\/sup><\/p>\n<\/td>\n

\n

3.83 \u00b1
1.06***<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Working memory – Number of entries<\/strong> – Y-maze<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

3.67 \u00b1
0.45<\/p>\n<\/td>\n

\n

3.00 \u00b1
0.30<\/p>\n<\/td>\n

\n

2.33 \u00b1
0.22<\/p>\n<\/td>\n

\n

2.83 \u00b1
0.27<\/p>\n<\/td>\n

\n

2.83 \u00b1
0.37<\/p>\n<\/td>\n

\n

3.33 \u00b1
0.81<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

2.33 \u00b1
0.22<\/p>\n<\/td>\n

\n

3.00 \u00b1
0.30<\/p>\n<\/td>\n

\n

1.17 \u00b1
0.11***<\/sup><\/p>\n<\/td>\n

\n

2.50 \u00b1
0.38<\/p>\n<\/td>\n

\n

1.50 \u00b1
0.23**<\/sup><\/p>\n<\/td>\n

\n

2.17 \u00b1
0.32<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

1.83 \u00b1
0.21<\/p>\n<\/td>\n

\n

3.50 \u00b1
0.15###<\/sup><\/p>\n<\/td>\n

\n

2.17 \u00b1
0.32**<\/sup><\/p>\n<\/td>\n

\n

2.50 \u00b1
0.15***<\/sup><\/p>\n<\/td>\n

\n

1.67 \u00b1
0.22***<\/sup><\/p>\n<\/td>\n

\n

1.83 \u00b1
0.21***<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

2.33 \u00b1
0.28<\/p>\n<\/td>\n

\n

3.83 \u00b1
0.11##<\/sup><\/p>\n<\/td>\n

\n

2.50 \u00b1
0.62<\/p>\n<\/td>\n

\n

2.33 \u00b1
0.28**<\/sup><\/p>\n<\/td>\n

\n

2.50 \u00b1
0.34*<\/sup><\/p>\n<\/td>\n

\n

3.00 \u00b1
0.63<\/p>\n<\/td>\n<\/tr>\n

\n

Working memory – Time spent (s)<\/strong> – Y-maze<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

66.83 \u00b1
10.41<\/p>\n<\/td>\n

\n

96.17 \u00b1
17.07<\/p>\n<\/td>\n

\n

88.33 \u00b1
8.38<\/p>\n<\/td>\n

\n

101.00 \u00b1
16.16<\/p>\n<\/td>\n

\n

91.50 \u00b1
10.25<\/p>\n<\/td>\n

\n

86.67 \u00b1
8.93<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

38.50 \u00b1
8.96<\/p>\n<\/td>\n

\n

54.50 \u00b1
7.63<\/p>\n<\/td>\n

\n

34.67 \u00b1
4.78<\/p>\n<\/td>\n

\n

39.33 \u00b1
6.27<\/p>\n<\/td>\n

\n

62.33 \u00b1
17.32<\/p>\n<\/td>\n

\n

58.00 \u00b1
11.22<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

30.50 \u00b1
4.33<\/p>\n<\/td>\n

\n

50.50 \u00b1
3.68##<\/sup><\/p>\n<\/td>\n

\n

28.67 \u00b1
2.02***<\/sup><\/p>\n<\/td>\n

\n

28.83 \u00b1
3.31**<\/sup><\/p>\n<\/td>\n

\n

18.17 \u00b1
2.08***<\/sup><\/p>\n<\/td>\n

\n

44.33 \u00b1
8.28<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

32.00 \u00b1
4.13<\/p>\n<\/td>\n

\n

53.83 \u00b1
6.52<\/p>\n<\/td>\n

\n

16.50 \u00b1
3.87<\/p>\n<\/td>\n

\n

23.83 \u00b1
2.97<\/p>\n<\/td>\n

\n

29.50 \u00b1
1.72<\/p>\n<\/td>\n

\n

21.33 \u00b1
5.17<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

n = 12, Data as mean \u00b1 standard error of mean. Numbers in bracket indicate dose (microgram\/day intranasal). VC: vehicle control, SC: CUMS control, BUS- Buspirone, MBT -= Marble burying test, ## P < 0.01, ### P < 0.001 (vs. VC), * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. CS).<\/p>\n\n\n

Effect on Y maze Parameter<\/strong><\/p>\n\n\n\n

Two-way repeated-measures ANOVA of\nworking memory-related data on number of entries into novel arm (Table 1)\nshowed significant effects with respect to time (F(2.561, 197.2) = 8.977, P\n< 0.001) and treatments (F(6, 77) = 4.533, P < 0.001). Dunnett’s test\nshowed no significant effects of treatment between the groups on D0 (SC vs VC,\nBUS or INDCA-NS vs. SC). Number of entries in SC group (vs\/ VC) was significant\n(P < 0.001 or P < 0.001) on D42 and D49, respectively; however, the\ndifference between SC and VC was not statistically significant on D35. On D42,\nthe number of entries in the BUS- and INDCA-NS (all doses)-treated groups was\nsignificantly lower. On D49, INDCA-NS (10 and 30)-treated groups showed\nsignificantly fewer entries (vs. SC), whereas the BUS- and INDCA-NS\n(100)-treated groups showed no significant changes (vs. SC). <\/p>\n\n\n\n

Two-way repeated-measures ANOVA of time spent in the novel arm (Table 1) showed significant effects with respect to time (F(2.015, 155.1) = 70.95, P < 0.001)  or treatment (F(6, 77) = 2.488, P < 0.05). Dunnett’s test revealed no significant effects of treatments, on D0, D35 or D49, between the groups (SC vs VC, BUS or INDCA-NS vs. SC). On D42, time spent signing was higher in SC (vs. VC) and lower in BUS and INDCA-NS (10 and 30), but not in INDCA-NS (100).<\/p>\n\n\n\n

Effect on MWM Parameters<\/strong><\/p>\n\n\n\n

Two-way ANOVA of escape latency data from\nthe MWM (Table 2)\nindicated significant effects of or treatment (F(6, 77) = 6.215, P < 0.001)\nor time (F(3, 231) = 70.84, P < 0.001) Dunnett’s test on D0, showed no\neffects in SC (vs VC) and BUS or INDCA-NS treated groups (vs SC) indicating\nuniform groupings. SC showed a significant increase in escape latencies compared\nwith VC on D35, D42, and D49. Significant prevention of SC-induced enhanced\nescape latencies (vs. SC) was observed in the BUS (P < 0.05) and INDCA-NS\ngroups at all tested doses on D49 but not on D35 or D42. <\/p>\n\n\n\n

The ANOVA of time data spent near the\ntarget zone from the MWM indicated significant effects of time (F(2.409, 185.5)\n= 32.65, P < 0.001), but not of treatment (F(6, 77) = 1.877, not\nsignificant). In addition, none of treatments showed significant differences\nbetween groups on any of days.<\/p>\n\n\n\n

Two-way ANOVA of the total distance\ntravelled (path length) from the MWM indicated significant effects of treatment\n(F(6, 77) = 3.515, P < 0.01) or time (F(2.595, 199.8) = 58.84, P <\n0.001). Dunnett’s test on D0 showed no effects in SC (vs. VC) and BUS or INDCA-NS\ntreated groups (vs. SC), indicating uniform grouping. SC showed a significant\nincrease in the total distance travelled on D35 (P < 0.01) and D42 (P <\n0.05), but not on D49 (vs. VC). None of the treatments prevented SC-induced\nincrease in total distance travelled on the day compared to SC.  <\/p>\n\n\n\n

Table 2: Effects of intermediate to long-term symptoms of CUMS<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Day<\/strong><\/p>\n<\/td>\n

\n

VC<\/strong><\/p>\n<\/td>\n

\n

SC<\/strong><\/p>\n<\/td>\n

\n

BUS<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(10)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(30)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(100)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Spatial memory – Escape latency (s) – MWM<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

7.58 \u00b1
1.18<\/p>\n<\/td>\n

\n

8.00 \u00b1
1.80<\/p>\n<\/td>\n

\n

7.00 \u00b1
1.40<\/p>\n<\/td>\n

\n

6.92 \u00b1
1.67<\/p>\n<\/td>\n

\n

8.92 \u00b1
1.70<\/p>\n<\/td>\n

\n

6.92 \u00b1
1.42<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

7.67 \u00b1
0.92<\/p>\n<\/td>\n

\n

35.92 \u00b1
5.49###<\/sup><\/p>\n<\/td>\n

\n

40.83 \u00b1
5.59<\/p>\n<\/td>\n

\n

42.25 \u00b1
4.75<\/p>\n<\/td>\n

\n

43.17 \u00b1
5.25<\/p>\n<\/td>\n

\n

36.83 \u00b1
5.29<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

8.00 \u00b1
1.04<\/p>\n<\/td>\n

\n

27.33 \u00b1
4.57##<\/sup><\/p>\n<\/td>\n

\n

20.58 \u00b1
5.06<\/p>\n<\/td>\n

\n

23.17 \u00b1
4.88<\/p>\n<\/td>\n

\n

25.17 \u00b1
4.33<\/p>\n<\/td>\n

\n

22.5 \u00b1
4.66<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

7.58 \u00b1
1.29<\/p>\n<\/td>\n

\n

32.25 \u00b1
3.90###<\/sup><\/p>\n<\/td>\n

\n

17.00 \u00b1
3.10*<\/p>\n<\/td>\n

\n

12.75 \u00b1
4.25**<\/p>\n<\/td>\n

\n

11.75 \u00b1
1.91***<\/p>\n<\/td>\n

\n

9.33 \u00b1
1.86***<\/p>\n<\/td>\n<\/tr>\n

\n

Spatial memory – Time spend near target zone (s) – MWM<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

4.5 \u00b1
1.18<\/p>\n<\/td>\n

\n

2.5 \u00b1
0.34<\/p>\n<\/td>\n

\n

3.42 \u00b1
0.75<\/p>\n<\/td>\n

\n

2.5 \u00b1
0.67<\/p>\n<\/td>\n

\n

3.33 \u00b1
0.59<\/p>\n<\/td>\n

\n

3.25 \u00b1
0.59<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

4.42 \u00b1
0.69<\/p>\n<\/td>\n

\n

11.25 \u00b1
2.99<\/p>\n<\/td>\n

\n

11.67 \u00b1
0.88<\/p>\n<\/td>\n

\n

9.75 \u00b1
1.35<\/p>\n<\/td>\n

\n

6.58 \u00b1
1.28<\/p>\n<\/td>\n

\n

11.25 \u00b1
2.48<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

4.67 \u00b1
0.86<\/p>\n<\/td>\n

\n

8.75 \u00b1
1.61<\/p>\n<\/td>\n

\n

9.17 \u00b1
1.54<\/p>\n<\/td>\n

\n

11.75 \u00b1
3.14<\/p>\n<\/td>\n

\n

11.42 \u00b1
1.59<\/p>\n<\/td>\n

\n

11.00 \u00b1
2.41<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

4.00 \u00b1
1.01<\/p>\n<\/td>\n

\n

5.92 \u00b1
0.92<\/p>\n<\/td>\n

\n

6.83 \u00b1
1.58<\/p>\n<\/td>\n

\n

4.00 \u00b1
0.83<\/p>\n<\/td>\n

\n

3.75 \u00b1
0.73<\/p>\n<\/td>\n

\n

4.25 \u00b1
0.80<\/p>\n<\/td>\n<\/tr>\n

\n

Spatial Memory – Total distance travelled (cm) – MWM<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

158.47 \u00b1
21.72<\/p>\n<\/td>\n

\n

143.7 \u00b1
19.01<\/p>\n<\/td>\n

\n

240.34 \u00b1
46.82<\/p>\n<\/td>\n

\n

190.7 \u00b1
50.98<\/p>\n<\/td>\n

\n

251.49 \u00b1
53.70<\/p>\n<\/td>\n

\n

169.99 \u00b1
26.29<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

181.08 \u00b1
25.26<\/p>\n<\/td>\n

\n

802.83 \u00b1
137.10##<\/sup><\/p>\n<\/td>\n

\n

843.33 \u00b1
137.68<\/p>\n<\/td>\n

\n

904.33 \u00b1
94.78<\/p>\n<\/td>\n

\n

714.33 \u00b1
138.65<\/p>\n<\/td>\n

\n

729.08 \u00b1
109.71<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

171.92 \u00b1
24.84<\/p>\n<\/td>\n

\n

422.83 \u00b1
75.66#<\/p>\n<\/td>\n

\n

366.58 \u00b1
106.52<\/p>\n<\/td>\n

\n

446.58 \u00b1
87.71<\/p>\n<\/td>\n

\n

511.58 \u00b1
92.11<\/p>\n<\/td>\n

\n

423.58 \u00b1
90.02<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

193.00 \u00b1
36.65<\/p>\n<\/td>\n

\n

448.42 \u00b1
104.43<\/p>\n<\/td>\n

\n

336.67 \u00b1
122.11<\/p>\n<\/td>\n

\n

155.08 \u00b1
51.86<\/p>\n<\/td>\n

\n

310.42 \u00b1
56.57<\/p>\n<\/td>\n

\n

255.5 \u00b1
50.61<\/p>\n<\/td>\n<\/tr>\n

\n

Anhedonia – % Sucrose consumption – SPT<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

51.78 \u00b1
5.28<\/p>\n<\/td>\n

\n

47.88 \u00b1
8.39<\/p>\n<\/td>\n

\n

59.53 \u00b1
7.60<\/p>\n<\/td>\n

\n

28.50 \u00b1
5.12<\/p>\n<\/td>\n

\n

35.81 \u00b1
7.53<\/p>\n<\/td>\n

\n

32.49 \u00b1
7.60<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

60.03 \u00b1
7.02<\/p>\n<\/td>\n

\n

57.07 \u00b1
2.31<\/p>\n<\/td>\n

\n

64.81 \u00b1
6.05<\/p>\n<\/td>\n

\n

50.83 \u00b1
7.49<\/p>\n<\/td>\n

\n

55.29 \u00b1
6.38<\/p>\n<\/td>\n

\n

41.30 \u00b1
6.18<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

63.66 \u00b1
6.95<\/p>\n<\/td>\n

\n

34.35 \u00b1
5.07#<\/sup><\/p>\n<\/td>\n

\n

70.12 \u00b1
5.63***<\/p>\n<\/td>\n

\n

71.21 \u00b1
3.65***<\/p>\n<\/td>\n

\n

66.46 \u00b1
5.44**<\/p>\n<\/td>\n

\n

59.48 \u00b1
4.09**<\/p>\n<\/td>\n<\/tr>\n

\n

Aggression – Attack latency (s) – RIT <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

D0<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n<\/tr>\n

\n

D35<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

142.25 \u00b1
27.03###<\/sup><\/p>\n<\/td>\n

\n

112.00 \u00b1
28.59<\/p>\n<\/td>\n

\n

136.83 \u00b1
19.88<\/p>\n<\/td>\n

\n

116.92 \u00b1
18.88<\/p>\n<\/td>\n

\n

80.42 \u00b1
18.61<\/p>\n<\/td>\n<\/tr>\n

\n

D42<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

123.08 \u00b1
25.14###<\/sup><\/p>\n<\/td>\n

\n

261.25 \u00b1
10.99***<\/p>\n<\/td>\n

\n

261.83 \u00b1
10.37***<\/p>\n<\/td>\n

\n

262.00 \u00b1
11.59***<\/p>\n<\/td>\n

\n

269.17 \u00b1
10.91***<\/p>\n<\/td>\n<\/tr>\n

\n

D49<\/p>\n<\/td>\n

\n

300.00 \u00b1
0.00<\/p>\n<\/td>\n

\n

78.25 \u00b1
18.16###<\/sup><\/p>\n<\/td>\n

\n

294.42 \u00b1
2.20***<\/p>\n<\/td>\n

\n

288.58 \u00b1
4.53***<\/p>\n<\/td>\n

\n

296.83 \u00b1
1.52***<\/p>\n<\/td>\n

\n

297.67 \u00b1
1.34***<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

n = 12, Data as mean \u00b1 standard error of mean. Numbers in bracket indicate dose (microgram\/day\/rat intranasal). VC: vehicle control, SC: CUMS control, SPT: Sucrose preference test, BUS: buspirone, MWM: Morris water maze, RIT – Resident intruder test. # P < 0.05, ## P < 0.01, ### P < 0.001 (vs. VC), * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. CS).<\/p>\n\n\n

Effect on SPT Parameters<\/strong><\/p>\n\n\n\n

Sucrose consumption data during SPT, as shown in Table 2, indicated significant effects of treatment and time (F(6, 77) = 3.09, P < 0.01) and (F(1.912, 147.2) = 20.87, P < 0.001). Dunnett’s test showed a significant decrease in % sucrose consumption in SC (vs. VC) on D49, but not on D35 or D42. Treatment with BUS and INDCA-NS (all tested doses) significantly prevented the SC-induced decrease in % sucrose consumption on D49, but not on D35 or D42 (vs. SC). <\/p>\n\n\n\n

Effect on RIT Parameters<\/strong><\/p>\n\n\n\n

Two-way ANOVA of the attack latency data (Table 2) indicated significant effects of time and treatment (F(1.805, 139.0) = 267.5, P < 0.001) and (F(6, 77) = 20.41, P < 0.01), respectively. Dunnett’s test showed significant (P < 0.001) reduction in attack latencies in SC (vs VC) on D35, D42 and D49. The BUS- and INDCA-NS (all tested doses)-treated groups showed significant (P < 0.001) prevention of SC-induced decline in attack latencies on D42 and D49, but not on D35. <\/p>\n\n\n\n

Effect on Hypothalamic Cortisol<\/strong><\/p>\n\n\n\n

One-way ANOVA of cortisol level data (Table 3) showed significant effects (F(6, 21) = 24.10, P < 0.001). SC showed higher cortisol levels than those in the VC (P < 0.001), whereas BUS and INDCA-NS (30 and 100), but not INDCA-NS (10), significantly prevented SC-induced increases in cortisol levels. <\/p>\n\n\n\n

Effect on Hypothalamic BDNF <\/strong><\/p>\n\n\n\n

One-way ANOVA of BDNF levels (Table 3)\nshowed significant treatment effects (F(6, 49) = 2.938, P < 0.05). BDNF\nlevels in the SC were higher, but not significantly different (vs. VC). In\naddition, BDNF levels in the BUS or INDCA-NS groups (at the tested doses) did\nnot show a significant difference between the groups (vs. SC). <\/p>\n\n\n\n

Table 3: Effects on biochemical markers of CUMS<\/strong><\/p>\n\n\n\n\n\n\n
\n

Measure<\/strong><\/p>\n<\/td>\n

\n

VC<\/strong><\/p>\n<\/td>\n

\n

SC<\/strong><\/p>\n<\/td>\n

\n

BUS<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(10)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(30)<\/strong><\/p>\n<\/td>\n

\n

INDCA-<\/strong>
NS(100)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Cortisol
(ng\/g)<\/p>\n<\/td>\n

\n

0.44
\u00b1 0.17<\/p>\n<\/td>\n

\n

38.93
\u00b1 3.75###<\/sup><\/p>\n<\/td>\n

\n

19.82
\u00b1 5.24**<\/sup><\/p>\n<\/td>\n

\n

30.31
\u00b1 4.25<\/p>\n<\/td>\n

\n

1.01
\u00b1 0.33***<\/sup><\/p>\n<\/td>\n

\n

0.66
\u00b1 0.25***<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

BDNF
(pg\/g)<\/p>\n<\/td>\n

\n

204.60
\u00b1 8.36<\/p>\n<\/td>\n

\n

311.50
\u00b1 21.42<\/p>\n<\/td>\n

\n

325.60
\u00b1 32.82<\/p>\n<\/td>\n

\n

356.30
\u00b1 39.75<\/p>\n<\/td>\n

\n

268.70
\u00b1 49.15<\/p>\n<\/td>\n

\n

213.70
\u00b1 30.36<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

n = 12, Data as mean \u00b1 standard error of mean. Numbers in bracket indicate dose (microgram\/day\/rat intranasal).VC \u2013 Vehicle control, SC- CUMS Stress control, BUS- Buspirone, # P < 0.05, ## P < 0.01, ### P < 0.001 (vs VC), * P < 0.05, ** P < 0.01, *** P < 0.001 (vs CS),<\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The effects of INDCA-NS on early and\nshort-term (anxiety and working memory) and intermediate-to long-term (spatial\nmemory, anhedonia, and aggression) changes in CUMS-induced chronic stress in\nrats were explored. Subacute administration of INDCA-NS resulted in consistent\nand dose-dependent prevention of CUMS-induced anxiety, anhedonia, and\naggression, but not cognitive decline, that is, loss of working and spatial\nmemory. These findings are consistent with CUMS-preventive effects observed in\nrats treated with BUS, a serotonergic agent, suggesting a serotonergic\nmechanism of INDCA-NS. The findings of previous studies on the oral\nadministration of INDCA to laboratory rats have provided further support27<\/sup>. <\/p>\n\n\n\n

Recently, triterpenoids containing CA\nextracts (unstandardized form) showed stress-relieving efficacy in animal\nmodels of chronic stress in zebrafish24<\/sup> and\nCUMS induced rats46<\/sup>. The present results are not only in line with these reports, but\nalso extend to a triterpenoid-based standardized version of INDCA-NS with\nintranasal administration against chronic stress-related symptoms. <\/p>\n\n\n\n

The CUMS paradigm was used to mimic\neveryday stress (unpredictable) and its effects on psychological well-being in\na reliable and clinically validated manner in rats47, 48<\/sup>.\nThese stressors included social stressors (isolation, cage mate changes, and\ncrowding), environmental stressors (cage tilting, damp bedding, and altered\nlight\/dark cycles), and physiological stressors (food\/water deprivation and\nrestraint), which are presented at random intervals and durations48, 49<\/sup>.\nBecause CUMS protocol prevents habituation (limitation of acute stress), it\nproduces clinically relevant behavioral changes, such as anxiety, depression,\ncognitive decline, anadenia, and aggression associated with psychological\nstress48, 50, 51<\/sup>. <\/p>\n\n\n\n

The efficacy of triterpenoids of INDCA-NS\nis suggested to involve serotonergic receptors27<\/sup> for efficacy against stress disorders, including  depression and anxiety26, 52<\/sup>. Concurrently, the role of\nserotoninergic agonist action has been confirmed in the mechanism of buspirone,\na drug with anxiety prophylaxis53<\/sup> and\nmanagement of stress-induced disorders54<\/sup>. Therefore, BUS was used as a positive control. <\/p>\n\n\n\n

In current\ninvestigation, CUMS for 35 days induced anxiety-like\nbehavior during marble MBT, with an increased number of buried marbles, in\nagreement with previous reports55<\/sup>. MBT has been extensively used in rodents to model anxiety and\nother stress-related behaviors56, 57<\/sup>.\nThese anxiety-like symptoms have been reported as early or intermediate\nfeatures of CUMS-induced pathology50<\/sup>. The timelines of anxiety in CUMS align with clinical observations,\nindicating suitability of CUMS for modeling the complex interplay between\nanxiety and depression50<\/sup>. Subacute INDCA-NS dose-dependently prevented the stress-induced\nincrease in the number of buried marbles, indicating its anxiolytic efficacy\nagainst CUMS.<\/p>\n\n\n\n

Short-term spatial and working memory\ndeficits are crucial components of executive function and have been linked to\nvarious neurodegenerative and psychiatric disorders. The Y-maze test is often\nutilized to evaluate the effects of treatments on working memory58<\/sup>. Chronic stress impairs spatial recognition memory, as evidenced by\ndecreased performance in the Y-maze test59<\/sup>. In the present study, a higher number of entries and time in novel\narms indicated working memory deficit in SC group (because of CUMS), whereas\nsignificant prevention of SC-induced increase in Y-maze parameters indicated\nprotection from working memory deficit by subacute nasal administration of\nINDCA-NS. <\/p>\n\n\n\n

In this study, the effects on spatial\nmemory were assessed using the MWM, which is the most reliable and widely\naccepted tool60<\/sup>. The increased escape latency, time spent near the target zone, and\ndecreased distance travelled during MWM in the SC group indicated impaired\nlong-term spatial memory with CUMS, which is in line with earlier reports50<\/sup>. However, INDCA-NS and BUS did not show any preventive effects on\nspatial memory decline in this study. <\/p>\n\n\n\n

Subacute intranasal INDCA-NS and BUS\nreversed the CUMS-induced decrease in sucrose consumption during the SPT on D49\nin a dose-dependent manner. Stress-induced depression-like behavior and\nstructural plasticity in the brain are characteristic of CUMS model61<\/sup>. Moreover, SPT involves measuring the preference of animals for a\nsucrose solution over water (a pleasurable activity) and a reduced sucrose\npreference, indicating an anhedonia state, which is common in chronic\nstress-related disorders62<\/sup>. <\/p>\n\n\n\n

Previously, a correlation between\nserotonergic system enhancement and reversal of CUMS-induced anhedonia has been\nreported. For example, CUMS-induced anhedonia \ndecreases hippocampal serotonin transporter protein levels, whereas\nnon-anhedonia increases them63<\/sup>. In addition, the serotonergic agent fluoxetine has been reported\nto prevent CUMS-induced decreases in serotonin transporter levels63<\/sup>. Upon nasal treatment, INDCA-NS and BUS (5HT1A agonist) not only\nprevented CUMS-induced anhedonia but also increased sucrose preference compared\nto VC (without CUMS). The similarity in efficacy between INDCA-NS and BUS\nreveals the function of serotonergic mechanisms in INDCA-NS action against CUMS54, 64<\/sup>.<\/p>\n\n\n\n

Aggression is an intermediate-to\nlong-term symptom associated with chronic stress-related depression and is\ncorrelated with a decrease in serotonin and a rise in dopamine65<\/sup>. CUMS mimics many aspects of stress-related depression, including61<\/sup>. We used RIT, most acceptable measure of aggression66<\/sup>. CUMS induction increased attack latencies (increased aggression)\nin the SC group from D35, when intranasally presented with INDCA-NS and BUS. <\/p>\n\n\n\n

The role of the serotonergic system has\nbeen reported in stress-induced67<\/sup> and aggressive behaviors in rodents68<\/sup>. For examples: 5HT1A agonists (BUS) and  reuptake inhibitors (fluoxetine) have been\nreported to reverse isolation stress-induced aggression69<\/sup>. The role of serotonin receptors, especially 5-HT1A\/1 B receptors,\nin the anti-migraine mechanism of INDCA-NS has been reported27<\/sup>. These reports strongly indicate a role for serotonergic receptors\nin the anti-stress mechanism of CUMS. <\/p>\n\n\n\n

In present study, CUMS induction in rats of the CS group caused a significant elevation in cortisol in the hypothalamus, which was prevented by subacute intranasal INDCA-NS and BUS. Cortisol, a glucocorticoid hormone, has been proposed as a promising biomarker for chronic stress assessment17, 70<\/sup> because of activated HPA axis71<\/sup>. This notion is supported by involvement of HPA axis and elevated cortisol in the mechanism of action of INDCA-NS in stress-related neuropsychiatric conditions22<\/sup>.<\/p>\n\n\n\n

In recent years, chronic stress has been reported to decrease brain BDNF72<\/sup>. Stress is a significant contributor to depression, especially anhedonia73<\/sup>. Many antidepressants may exert their effects by regulating BDNF74<\/sup>. Moreover, BDNF plays the foremost role in HPA axis72<\/sup>. However, present study showed elevated levels of BDNF in the hypothalamus of CUMS-induced rats, although the difference was not significant. These effects are in agreement with previous reports of increased BDNF in the dorsal  and ventral hippocampi of CUMS-induced rats75<\/sup> and chronically restrained rats76<\/sup>. Moreover, INDCA-NS and BUS did not affect BDNF levels in CUMS-induced rats.<\/p>\n\n\n\n

Existing evidence indicates a strong relationship between BDNF and serotonin levels 77<\/sup> especially under chronic stress78<\/sup>. The enhanced BDNF levels (in the present study) and serotoninergic properties of INDCA-NS27<\/sup> indicate a synergistic role of two seemingly distinct signaling systems79<\/sup> in anti-stress mechanism of INDCA-NS. In the past, enhanced BDNF levels were reported to be involved in CA\u2019s neuroprotective80-82<\/sup>  and anti-stress83<\/sup> properties of CA.<\/p>\n\n\n\n

Conclusions<\/strong><\/p>\n\n\n\n

The intranasal subacute administration of INDCA-NS was effective in preventing early to short-term (anxiety and working memory) and intermediate to long-term (anhedonia and aggression) symptoms of CUMS in laboratory rats, probably through serotonin and cortisol involvement. The potential of INDCA-NS can be explored with suitable clinical studies for management stress-related disorders such as tension-type headache or migraine. <\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

The authors would like to acknowledge Dr. K.R. Mahadik, Ex-Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The study was supported by Indus Biotech Limited, Pune, India (Grant Project No: IBS432).<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The author(s) do not have any conflict of interest.<\/p>\n\n\n\n

Data Availability<\/strong><\/p>\n\n\n\n

The manuscript incorporates all datasets produced or examined throughout this research study<\/p>\n\n\n\n

Ethics Statement<\/strong><\/p>\n\n\n\n

The experimental protocols were sanctioned by \u201cInstitutional Animal\nEthics Committee\u201d (IAEC) of Poona College of Pharmacy, Bharati Vidyapeeth\nDeemed University, Pune, India (Approval number CPCSEA\/ PCP\/PCL33\/2018-19)<\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n\n\n\n

Clinical Trial Registration<\/strong><\/p>\n\n\n\n

This research does not involve any clinical trials<\/p>\n\n\n\n

Authors’ Contributions<\/strong><\/p>\n\n\n\n

Each author mentioned has significantly and directly contributed intellectually to the project and has given their approval for its publication. <\/strong><\/p>\n\n\n\n

Prasad Thakurdesai<\/strong>: Conceptualization, Methodology, Writing \u2013 review and editing, Visualization, Supervision, Funding acquisition; <\/p>\n\n\n\n

Gayatri Shivsingwale<\/strong>: Investigation, Formal analysis; <\/p>\n\n\n\n

Urmila Aswar<\/strong>: Methodology, Software, Validation, Formal analysis, Resources, Data curation, Writing \u2013 original draft, Writing \u2013 Review and editing, Supervision, Project administration.<\/p>\n\n\n\n

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