Neuroprotective Effects of Intranasally Administered Octadecaneuropeptide Analog in a Mouse Model of MPTP-Induced Parkinson's Disease

Summary

Intranasal administration enables direct delivery of bioactive molecules to the brain, providing a non-invasive alternative to the intracerebroventricular method. This study demonstrates that intranasal administration of cyclo_(1-8)OP, an ODN analog, exerts strong neuroprotective effects against MPTP-induced oxidative damage and apoptosis in the striatum in a mouse model of Parkinson's disease, highlighting therapeutic potential.

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder for which existing therapies are primarily palliative and lack curative efficacy. Octadecaneuropeptide (ODN) is a peptide exclusively produced by astrocytes that protects neurons against oxidative cell damage and apoptosis in in vitro and in vivo models of PD. However, ODN cannot cross the blood-brain barrier (BBB) and requires intracerebroventricular injection to reach the brain, presenting a significant challenge for therapeutic application. This study investigates the neuroprotective efficacy of cyclo_(1-8)OP, an ODN analog, delivered via the intranasal (IN) route to bypass the BBB in an in vivo model of PD. Male C57BL/6J mice were used for the experiments and divided into four groups: sham, MPTP- (20 mg/kg body weight, intraperitoneal), cyclo_(1-8)OP- (10 ng/10 µL, IN), and MPTP + cyclo_(1-8)OP-treated animals. On day 0 (D0), animals received three intraperitoneal injections of 100 µL MPTP solution at 2-h intervals (MPTP- and MPTP + cyclo_(1-8)OP-treated mice) or saline solution (sham and cyclo_(1-8)OP-treated mice). One hour after the final injection, 10 µL of IN instillation of cyclo_(1-8)OP (cyclo_(1-8)OP- and MPTP + cyclo_(1-8)OP-treated mice) or IN saline solution (sham and MPTP-treated mice) was administered. On D7, a cylinder test was performed to assess motor function. Subsequently, animals were sacrificed, and the striatum was removed and analyzed by RT-qPCR to assess caspase-3 gene expression. Tissue samples were also used to measure antioxidant enzyme activities, reactive oxygen species (ROS), malondialdehyde (MDA), and carbonylated protein abundance. A single IN dose of 10 ng cyclo_(1-8)OP, administered 1 h after the final MPTP dose, prevented neurotoxicity in the striatum 7 days post-treatment. Cyclo_(1-8)OP-mediated neuroprotection was associated with strong inhibition of caspase-3 expression induced by MPTP in the striatum. Additionally, cyclo_(1-8)OP restored the activities of antioxidant enzymes, preventing the accumulation of ROS, lipid peroxidation products, and protein carbonylation in the striatum.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder marked by the progressive loss of dopaminergic neurons in the substantia nigra, which innervates the striatum. This loss results in significant neurological symptoms, including severe motor deficits such as muscle rigidity, akinesia, and bradykinesia¹. The prevalence of neurodegenerative diseases like PD continues to rise, primarily due to an aging population, posing a substantial public health challenge². Current treatments for PD predominantly rely on dopaminergic analogs and intracerebral high-frequency stimulation^3,4. However, these therapies lack neuroprotective effects and fail to halt disease progression, underscoring the urgent need for innovative strategies to prevent neuronal loss, promote regeneration, and mitigate disease advancement⁵.

Octadecaneuropeptide (ODN) is a peptide derived from the proteolytic processing of the 86-amino acid precursor diazepam-binding inhibitor (DBI)⁶, expressed primarily by astroglial cells in the mammalian central nervous system. The evolutionary conservation of ODN's primary structure suggests its significant biological roles. ODN has been shown to regulate key functions such as food intake, sleep, aggression, and anxiety-related behaviors⁷. Beyond its regulatory role, extensive research has demonstrated the neuroprotective properties of ODN in various neurological disorders associated with neuronal degeneration. For instance, ODN protects dopaminergic neurons in a model of PD⁸, and reduces infarct size while enhancing functional recovery following stroke in rodents⁹. One of ODN's key features is its ability to counteract many deleterious processes activated during brain damage through its anti-apoptotic, anti-inflammatory, antioxidant, and immunomodulatory activities^10,11,12. Furthermore, ODN may act beyond the acute phase of stroke by promoting neurogenesis and neuronal plasticity⁹. For potential clinical applications of peptides or their analogs as therapeutic agents, choosing an optimal route of administration is crucial to prevent rapid degradation in the bloodstream and minimize the first-pass effect. Among various delivery strategies, intranasal (IN) administration has emerged as a promising non-invasive alternative, allowing direct delivery of certain bioactive molecules to the brain while bypassing the blood-brain barrier (BBB)^13,14,15. This route eliminates the need for invasive stereotaxic injections and enhances brain biodistribution compared to peripheral organs. For instance, pituitary adenylate cyclase-activating polypeptide (PACAP) has demonstrated greater efficacy in brain uptake and neuroprotection when delivered by IN instillation compared to intravenous injection, significantly reducing infarct volume and improving functional recovery in stroke models⁹.

Although IN administration is a minimally invasive and efficient technique for delivering therapeutic agents directly to the brain by bypassing the BBB, its efficacy remains dependent on several practical parameters, including dose and total volume administered. In rodents, the recommended volume typically ranges from 10 to 25 µL per nostril to avoid aspiration or overflow into the gastrointestinal tract^16,17. The efficiency of nasal uptake is also affected by the animal's size, nasal cavity surface area, mucociliary clearance, and compound properties such as lipophilicity and molecular weight^18,19. These constraints should be considered during protocol design to ensure reproducibility and translational relevance in preclinical models.

An in vivo study has shown that intracerebroventricular (ICV) injection of low doses of ODN conferred significant neuroprotective effects, particularly by preventing the degeneration of dopaminergic neurons in MPTP-treated mice⁸. The current study aims to evaluate the feasibility and efficacy of IN administration of the ODN analog, cyclo_(1-8)OP, as a non-invasive alternative to ICV injection for delivering neuroprotective peptide analog to the brain, specifically to determine whether this route can prevent dopaminergic neuronal loss and improve motor outcomes in MPTP-treated mice, a well-established model of PD.

Protocol

All experiments followed the American Veterinary Medical Association guidelines and were approved by the Medical Ethical Committee of the Pasteur Institute (approval number: FST/LNFP/Pro 160220). Ten-week-old C57BL/6J male mice were obtained from the Pasteur Institute of Tunis. Mice were housed in three per cage in a temperature-controlled room (21 °C ± 1 °C) with a 12-h light/dark cycle and provided free access to food and water for 1 week of acclimatization. Details of the reagents and equipment used are listed in the Table of Materials.

1. Treatment procedure

1. Divide animals into four groups: sham (n = 9), MPTP- (n = 9), cyclo_(1-8)OP- (n = 9), and MPTP + cyclo_(1-8)OP-treated (n = 9).
2. Administer three intraperitoneal (IP) injections of MPTP (20 mg/kg b.w.) in 100 µL of 0.9% NaCl at 2-h intervals on the first day of treatment (D0). 1 h after the final MPTP injection, administer 10 µL intranasally (IN) of cyclo_(1-8)OP (10 ng) or saline.
3. Inject control animals with saline instead of MPTP, with or without cyclo_(1-8)OP (Figure 1).
4. On the seventh day (D7), subject mice to the cylinder test. Then deeply anesthetize the mice using an IP injection of pentobarbital and sacrifice them (following institutionally approved protocols) to collect brains for real-time PCR and biochemical analyses²⁰ (Figure 2).

2. Injection procedure

1. Intraperitoneal injection
   1. Position the mouse carefully and immobilize its head during the procedure.
   2. Orient the mouse head-down to allow the organs to shift naturally with gravity, minimizing any contact with the needle.
   3. Insert the needle at a 45-degree angle into the lower abdomen.
   4. Administer three 100 µL injections on the same side, with a 2-h interval between each injection, all performed on the same day.
   5. Inject 0.9% saline for the sham and cyclo_(1-8)OP groups, or MPTP solution (20 mg/kg b.w.) for the MPTP and MPTP + cyclo_(1-8)OP groups.
2. Intranasal administration
   NOTE: The selected dose (10 ng) was based on a previous study demonstrating the neuroprotective efficacy of ICV administration of ODN⁸. The total volume of 10 µL and droplet size (5 µL per nostril) were chosen according to established IN administration protocols^18,21. This volume is well below the average capacity of the mouse nasal cavity (0.032 cm³), thereby minimizing the risk of nostril obstruction or asphyxiation and ensuring safe and efficient nasal absorption²².
   1. Firmly hold the mouse by the scruff of its neck, ensuring its nose is oriented upward to facilitate precise dosing while minimizing head movement.
   2. Invert the animal so that its ventral side faces the ceiling, with its neck parallel to the floor. Ensure the mouse's head remains securely immobilized, preventing vertical or horizontal movement.
   3. Align the pipette tip with the mouse's nostril and slowly depress the plunger to form a small droplet.
   4. Place a 5 µL (half of the volume) droplet near each of the mouse's nostrils.
   5. Allow the mouse to naturally inhale the solution.
   6. Repeat these steps to ensure consistent dosing, with 10 µL of cyclo_(1-8)OP (10 ng) administered for the MPTP + cyclo_(1-8)OP and cyclo_(1-8)OP groups.
   7. Carefully observe the mouse's mouth throughout the procedure. The presence of liquid in the mouth may indicate incomplete nasal absorption. This could result from improper droplet size, rapid administration, or insufficient inhalation, allowing the solution to pool in the nasopharynx and subsequently exit through the mouth.
   8. Minimize this risk by reducing droplet size if necessary and ensuring adequate time for inhalation before administering additional volumes.
   9. Avoid immediately administering an additional dose to prevent excessive volume.
   10. Document any misplaced doses and carefully note any expelled liquid.
   11. Ensure each mouse fully inhales both droplets (one per nostril) before proceeding with further administration.
   12. Confirm accurate targeting of the olfactory bulb by performing an IN injection of methylene blue solution.
   13. Evaluate the accuracy by visually inspecting the deposition and spread of the blue coloring.

3. Cylinder test

1. Use a transparent cylinder, 20 cm in height and 15 cm in diameter, placed at the center of the table.
2. Position three black cardboard panels around the cylinder (4-8 cm away) to minimize environmental lighting effects. Leave one side of the cylinder open for video recording.
3. Set the camera 40-60 cm from the cylinder to capture its full view.
4. Place the mouse into the cylinder and begin recording immediately for 5 min.
5. After each mouse completes the test, clean the cylinder with water, spray 70% ethanol to remove scents, and wipe it dry before testing the next mouse.
6. Playback the recording at half-speed to allow detailed observation.
7. Count each instance where the mouse lifts its paws against the cylinder wall to assess exploratory behavior²³.

4. Preparation of brain tissue extracts for biochemical experiments

1. Anesthetize animals with pentobarbital (40 mg/kg, IP) and then decapitate them (following institutionally approved protocols).
2. Dissect the striatum and homogenize in 2 mL of TBS per gram of tissue using lysis buffer (1% Triton X-100, 50 mM Tris-HCl, 10 mM EDTA).
3. Centrifuge homogenates at 14,000 × g for 15 min at 4 °C.
4. Measure protein concentration using the Biuret colorimetric determination kit and store aliquots at −20 °C for later analysis.

5. Measurement of intracellular ROS formation

1. Incubate brain tissue extract with 10 µM of DCFH₂-DA at 37 °C for 30 min in the dark.
2. Remove the supernatant and wash twice with PBS (0.1 M, pH 7.4, 37 °C).
3. Measure the fluorescence using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

6. Measurement of oxidative stress markers

1. Malondialdehyde
   1. Mix 20 µL of sample (~50 µg protein) with 125 µL of 20% TCA and 50 µL of 0.67% TBA.
   2. Incubate at 95 °C for 30 min, then cool rapidly at 4 °C.
   3. Add 800 µL of butanol, vortex, and centrifuge at 3000 × g for 10 min at 4 °C.
   4. Measure the absorbance at 532 nm and calculate MDA levels using the extinction coefficient (1.65 × 10⁵ M^-1cm^-1).
2. Protein carbonyl measurement
   1. Mix 200 µL of DNPH with 50 µL of protein sample and incubate for 1 h.
   2. Add 250 µL of 20% TCA and cool for 10 min on ice.
   3. Centrifuge at 3000 × g for 10 min at 4 °C and wash the pellet with 10% TCA, then with ethanol:ethyl acetate (v/v).
   4. Resuspend the pellet in 250 µL of 6 M guanidine hydrochloride and measure absorbance at 370 nm.
   5. Calculate PCO levels using the extinction coefficient (22,000 M^-1cm^-1)²⁴.

7. Measurement of antioxidant enzyme activities

1. Superoxide dismutase assay
   1. Incubate the sample (~50 µg) with bovine catalase (0.4 U/L), DL-epinephrine (5 mg/mL), and Na₂CO₃/NaHCO₃ buffer (62.5 mM, pH 10.2).
   2. Measure epinephrine oxidation at 480 nm using a spectrophotometer for 300 s at 60 s intervals²⁵.
2. Catalase assay
   1. Mix the sample with 90 mM H₂O₂ in PBS.
   2. Monitor the decrease in H₂O₂ absorbance at 240 nm for 3 min at 30 s intervals.
   3. Calculate CAT activity using the extinction coefficient (40 M^-1cm^-1).

8. Real - time PCR analysis

1. Extract total RNA from the mouse striatum using the acid guanidinium thiocyanate-phenol-chloroform method.
2. Treat RNA with DNase I to remove genomic DNA and measure RNA concentration at 260 nm.
3. Perform quantitative RT-PCR using 1 µg of total RNA with a one-step Q-RT PCR kit.
4. Amplify cDNA with SYBR Green PCR Master Mix and primers (Table 1).
5. Calculate relative cDNA levels using the 2^−ΔΔCt method with GAPDH as the internal control²⁶.

9. Statistical analysis

1. Perform statistical analyses using GraphPad software.
2. Use Student's t-test for single comparisons. For multiple comparisons, use one-way or two-way ANOVA followed by Bonferroni's post hoc test, as appropriate. In all cases, consider a P-value of 0.05 or less as statistically significant.

Results

After MPTP administration, mice displayed characteristic Parkinsonian symptoms, including an elevated and rigid Straub tail, piloerection, immobility, and postural abnormalities such as spinal curvature, confirming the successful establishment of the MPTP model (Figure 3). Verification of IN delivery using methylene blue, with mice sacrificed 10 min post-injection, showed effective dye distribution within the targeted olfactory bulb and brain regions, supporting the precisio...

Discussion

Previous studies demonstrated that ICV administration of ODN counteracted MPTP-induced degeneration of nigrostriatal dopaminergic neurons, oxidative damage, and neuroinflammation⁸. The current investigation assessed the efficacy of IN delivery of the ODN analog, cyclo_(1-8)OP, for preventing MPTP-induced motor impairments, oxidative damage, and neurotoxicity in an in vivo PD model using C57BL/6J mice.

The results demonstrated that MPTP-treated mice ex...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2', 7'-Dichlorofluorescin DCFH2-DA	Sigma Aldrich	064M4008V
Bio-Rad spectrophotometer	Bio-Rad Laboratories, Philadelphia, PA, USA
Butanol
Camera
Catalase from bovine liver	SIGMA life science	SLBZ7596
cyclo(1-8)OP	INSERM U1239 NorDiC, Rouen
Cylinder test (graduated beaker )
DNPH
EDTA plus	Thermo Scientic	11836714
Ethanol 70%
Ethanol-ethylacetate solution
GraphPad software	La Jolla, CA, USA
Guanidine hydrochloride	GeneON	5CCAE310
H2O2	Pharmaghreb	AEQ10101-D
Insulin syringe 2ml
L-epinephrine	MP Biomedicals	151065
Methylene blue	Thermo Scientific Chemicals	10455081
Micropipette
Microplate reader Synergy LX-Agilent	Synergy LX-Agilent
MPTP	MedChemExpress	HY-15608
MPTP
Na2CO3/NaHCO3 buffer
NaCl 0.9
one-step Q-RT PCR kit	New England Biolabs
Pentobarbital	Sigma Aldrich
Synergy LX Nanodrop 2000 spectrophotometer	Biotek Agilent
Thiobarbituric acid (TBA, 0.67%)	LOBA CHEMIE PVT.LTD	626500025
Tips
Trichloroacetic acid (TCA, 20%)	Sisco Research laboratories SRL	6740608
Tri-Reagent	Sigma Aldrich	MFCD00213058
Tris-HCl	Thermo Scientic	J22638.K2
Triton X-100	Thermo Scientic	A16046.AE