Main Research Findings

1) When administered to rats, DSIP was found to recover motor function following experimentally-induced strokes.

2) DSIP was shown to promote stress protective and antioxidant actions as well as enhance the efficiency of oxidative phosphorylation.

 

Selected Data

1) The study conducted by researchers Tukhovskaya et al aimed to investigate the neuroprotective and motor function recovery effects of intranasal Delta Sleep-Inducing Peptide (DSIP) in Sprague-Dawley (SD) rats subjected to focal stroke induced by middle cerebral artery occlusion (MCAO). The experimental design included precise animal handling, MCAO surgical procedures, DSIP administration, and comprehensive behavioral and histological assessments [1].

Adult male SD rats, weighing between 320–380 g, were utilized. Rats were housed under a 12-hour light/dark cycle with unlimited access to food and water. A total of 26 male SD rats underwent the MCAO procedure to induce focal stroke. Animals were anesthetized intramuscularly with a ketamine–xylazine mixture composed of 30 mg/kg ketamine and 12 mg/kg xylazine, respectively, with a total injection volume of 1.2 mL/kg. The right temporal muscle was dissected, the temporal bone dried, and a laser Doppler flow (LDF) probe was attached to the temporal bone to monitor regional cerebral blood flow (rCBF) during the procedure. A midline cervical incision exposed the right common carotid artery (CCA) [1].

Loose ligatures were placed on the CCA and internal carotid artery (ICA). Two more loose ligatures were placed on the external carotid artery (ECA) at a point 2–3 mm distal from the CCA bifurcation. The occipital artery and the pterygopalatine artery (PPA) were permanently ligated. A silicon–Teflon monofilament 0.13 mm diameter with a 0.3 mm diameter heat-blunted end was used as the occluding suture. This suture was coated with 0.5% (w/v) poly-L-lysine solution and dried before use. The intraluminal suture was inserted through a small puncture opening in the ECA and advanced into the ICA by approximately 18 mm (adjusted for rat weight) until a slight resistance was felt.

Ischemia was confirmed by an rCBF decrease of more than 70% compared to baseline. The occlusion suture was left in place for 90 minutes. After 90 minutes, the suture was withdrawn, and successful reperfusion was confirmed by rCBF restitution to 90–120% of baseline within 10 minutes. The ligatures on the CCA, ICA, and PPA were removed, but the ECA was permanently ligated above and below the opening. The LDF probe was detached, and wounds were closed with a twisted suture. Sham-operated rats underwent the same surgical procedure except for the MCA occlusion. Rectal temperature was maintained at 37 °C using a heating pad until the animals recovered from anesthesia. Post-surgery, rats were returned to cages with free access to food and water [1].

The animals were divided into three main groups defined as follows: MCAO + Vehicle included animals subjected to MCAO and treated with vehicle (saline); MCAO + DSIP included animals subjected to MCAO and treated with DSIP; and sham-operated animals (no MCAO). DSIP or vehicle was administered intranasally at a dosage of 120 µg/kg (n a volume of 100 μL/kg. The treatment regimen included a preventive dose 60 ± 15 minutes prior to MCAO, followed by daily doses for 7 days after reperfusion.

A battery of behavioral tests was performed on days 1, 3, 5, 7, 14, and 21 after MCAO to assess motor deficits. All testing was conducted during the animals’ light cycle. The rotarod test was a quantitative assessment of motor coordination and balance using an automated rotarod. Animals were trained for 3 consecutive days prior to MCAO, with three trials per day. Each trial involved placing the animal on a rotarod revolving at 5 rpm/min, then accelerating at 0.6 rpm/min. Fall latency, defined as the time until the animal fell off, was measured. Animals were pre-selected into groups to ensure homogeneous performance. Latency times were recorded, with 0 seconds assigned if the animal was unable to grasp the rod [1].

The ketamine-induced rotation asymmetry test assessed drug-induced rotation asymmetry manually for 5 minutes after an intramuscular ketamine injection of 10 mg/kg. The percentage of rotations to the right (ipsilateral to the injured hemisphere) relative to total rotations was recorded. This test is based on the principle that unilateral MCAO induces ipsilateral rotations under ketamine.

After 21 days, animals were euthanized by CO2 inhalation, and their brains were perfusion-fixed. Hearts were exposed, and ascending aortas were catheterized via the left ventricle. Blood was flushed out with 150 mL of saline with 100 U/L heparin at 30 mL/min, followed by perfusion with 3% formaldehyde and 150 mL of 1% glutaraldehyde solution. Brains were decapitated, removed from the skull, cryoprotected in graded sucrose buffered solutions of 10%, 20%, and 30%, and flash-frozen at -65°C using a cryostat. Coronal cryosections of 50 µm thickness at 1 mm intervals were obtained and stained with cresyl violet. Sections were scanned and processed using a 3D-reconstruction program. The volumes of both hemispheres were calculated using the Cavalieri method. Indirect lesion area was determined by subtracting the intact area of the ipsilateral hemisphere from the contralateral hemisphere. Lesion volume was expressed as a percentage of the contralateral hemisphere volume [1].

2) This study conducted by Khvatova et al investigated the effects of DSIP and its analogue Deltaran on the respiratory activity of rat brain mitochondria and brain homogenates, as well as their stress-protective potency under experimental hypoxia. The methods involved careful preparation of biological samples, polarographic measurements of oxygen consumption, and a model of experimental hypoxia [2].

The study utilized white non-thoroughbred male rats weighing 180-200g. DSIP was chemically synthesized using a solid-phase method, with its purity (98.5%) confirmed by HPLC, NMR, and MS data. Deltaran, a newly developed and registered stress-protective medicine for intranasal application, was provided as a lyophilized mixture of DSIP and glycine. For each experiment, two to three rats were decapitated. The combined brain tissue from these rats was then used to isolate mitochondria via differential centrifugation in a sucrose gradient. This procedure involved removing the supernatant and resuspending the mitochondrial pellet in an isolation buffer of 250 mM sucrose, 0.5 mM EDTA, and 10 mM Tris-HCl to achieve a final protein concentration of 20-25 mg/ml [2].

In parallel experiments, whole brains from two rats were rapidly removed and placed in ice-cold isolation buffer. The forebrain tissue was dissected, finely minced with scissors in a small portion of the buffer, and washed four times. The residue was then homogenized in suspension with isolation buffer (10% w/v) using a hand-held glass homogenizer. These homogenates were then used for respiration assays.

Oxygen uptake by isolated mitochondrial fractions or brain homogenates was measured polarographically using an open Clark platinum microelectrode. The respiration buffer contained 250 mM sucrose, 0.5 mM EDTA, 15 mM KCl, 20 mM KH2PO4, and 10 mM Tris-HCl. Mitochondrial suspension was added to the experimental chamber to achieve a final protein concentration of 2.5–2.7 mg/ml. For brain homogenates, 0.2 ml of the preparation was diluted to 1 ml by the respiration buffer. 15 mM potassium glutamate was used as the oxidized substrate. Respiration substrate ADP was added to the chamber. To allow uncoupled respiration, 40 µM DNP (2,4-dinitrophenol) was added. DSIP or Deltaran was dissolved in double distilled water and added directly to the polarographic cuvette. DSIP was tested at final peptide concentrations of 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸ M. For Deltaran, the tested suspension contained 10⁻⁶ M DSIP and 10⁻⁵ M Glycine [2].

Oxygen uptake rates were determined to calculate key parameters of oxidative phosphorylation, the parameters were defined as follows. V3: Rate of phosphorylated respiration (state III), representing maximal oxygen consumption rate in the presence of ADP. V4: Rate of uncoupled respiration (state IV), representing oxygen consumption rate after ADP has been consumed. VDNP: Rate of respiration in the presence of an uncoupler (DNP), indicating maximal electron transport capacity. RCR (Respiratory Control Ratio): Ratio of V3 to V4, a measure of coupling efficiency. ADP/O: Ratio of ADP molecules phosphorylated to oxygen atoms reduced, reflecting phosphorylation efficiency. ADP/t: Rate of ADP phosphorylation.

To investigate the stress-protective potency of DSIP, rats were subjected to experimental hypoxia. Hypoxia stress was modeled by placing rats into a flow pressure chamber for 15 minutes under 0.26 bar, which simulates an altitude of 10,000 m above ground level. This altitude was achieved gradually at a rate of 1200 m/s. The animals tolerated the manipulation well, with no reported cases of death. DSIP was dissolved in saline and injected intraperitoneally at a dose of 120 µg/kg, 20 minutes prior to placing the animal into the hypoxic chamber. After hypoxia, mitochondrial suspensions were isolated from the brains of these rats using the same technique as described above for polarographic measurements [2].

 

Discussion

1) The study by Tukhovskaya et al investigated the impact of intranasal DSIP on motor function recovery and brain infarction volume in SD rats subjected to focal stroke induced by MCAO. The results demonstrate that DSIP significantly accelerates the recovery of motor function, although its effect on brain infarct volume did not reach statistical significance.

One of the primary outcomes assessed was motor coordination and balance using the rotarod test. Animals treated with DSIP (MCAO + DSIP group) showed a significantly improved performance in the rotarod test compared to vehicle-treated MCAO animals. On day 7 of testing, motor coordination in the DSIP-treated group began to recover, a trend that continued through day 21. This progressive improvement was confirmed by repeated measures ANOVA. Linear regression analysis of the fall latency revealed positive dynamics only in animals receiving DSIP. The slope of the linear regression line for the DSIP-treated group was significantly different from zero, indicating a steady and significant increase in fall latency over the 21 days of testing. In contrast, the vehicle-treated MCAO group showed no significant change in fall latency over time. Sham-operated animals maintained stable high fall latencies. The improvement in fall latency in DSIP-treated animals was statistically significant relative to their performance on day 1 of testing, further supporting DSIP’s beneficial effect on motor recovery [1].

Figure 1: Changes in fall latency during the rotarod test across the three different experimental groups.

The study also evaluated drug-induced rotation asymmetry, a measure of neurological deficit after unilateral MCAO. The results indicated that DSIP did not have a significant influence on rotation asymmetry compared to vehicle-treated animals. This suggests that while DSIP improves motor coordination, it may not directly ameliorate the specific asymmetry in motor behavior induced by ketamine in this stroke model. Brain infarct volume was quantified at 21 days post-MCAO. The mean brain infarction volume in the MCAO + DSIP group was 20.9 ± 6.9% of the contralateral hemisphere volume. This was numerically smaller than the MCAO + Vehicle group, which had an infarct volume of 24.1 ± 4.6%. Despite this numerical difference, the comparison between the DSIP-treated and vehicle-treated MCAO groups did not yield statistical significance. This indicates that while DSIP may have a tendency to reduce infarct size, the effect was not robust enough to be considered statistically significant under the experimental conditions. This result contrasts with some previous studies on DSIP’s neuroprotective effects that have reported reduction in infarct size in other models [1].

Figure 2: Changes in rotation asymmetry expressed as the percentage of the animal’s rotation in the direction of the damage relative to rotations in both directions.

The researchers concluded that intranasal administration of DSIP, given both preventively and therapeutically over 8 days, leads to accelerated recovery of motor functions in SD rats after focal stroke. This positive effect on motor performance, particularly evident in the rotarod test, highlights DSIP’s potential in mitigating post-stroke motor deficits. The lack of statistically significant reduction in infarct volume, despite a numerical decrease, suggests that DSIP’s primary benefit in this model might be more related to functional neurorecovery and neural network plasticity rather than a direct, massive reduction of necrotic tissue [1].

The discussion considers several potential mechanisms for DSIP’s observed benefits suggesting DSIP could be associated with the rescuing of neurons in motor cortex and subcortical structures involved in motor control. Previous research suggests DSIP can influence biosynthetic processes in the brain, reduce stress protein factors, and potentially affect glutamate and GABA receptors in regions like the cortex, thalamus, and hippocampus. Furthermore, DSIP is known to improve mitochondrial respiratory activity and enhance the efficiency of the enzymatic energy system. Previous work shows that DSIP can inhibit hypoxia-induced reduction of mitochondrial respiratory activity and suggests that DSIP’s antioxidant mechanisms could contribute to its neuroprotective effects.

The combined preventive and therapeutic administration strategy used in this study appears to have successfully avoided some of the negative consequences of DSIP administration during the occlusion period, which might have occurred with different timing or dosages. Overall, the study strongly supports DSIP as a promising therapeutic agent for functional recovery after ischemic stroke [1].

2) The results of the study performed by Khvatova et al demonstrate that DSIP significantly enhances the efficiency of oxidative phosphorylation in rat brain mitochondria and brain homogenates, and provides complete protection against hypoxia-induced reductions in mitochondrial respiratory activity.

The experiments first assessed the direct impact of DSIP on freshly isolated rat brain mitochondria and brain homogenates under normal (non-hypoxic) conditions using polarographic measurements of oxygen consumption. DSIP significantly increased the rate of phosphorylated respiration (V3), which represents the maximal oxygen consumption rate in the presence of ADP. This effect was observed across DSIP concentrations ranging from 10⁻⁵ to 10⁻⁷ M, showing a 10-20% elevation. Importantly, DSIP did not alter the rate of uncoupled respiration (VDNP), which is the oxygen consumption rate when the proton gradient is dissipated. This indicates that DSIP specifically improves ATP synthesis efficiency rather than simply increasing overall oxygen consumption or causing uncoupling [2].

DSIP was shown to significantly increase the rate of ADP phosphorylation (ADP/t) by 10-30%. The most effective concentration for this effect was 10⁻⁷ M, although a clear concentration dependence was not strictly observed. The respiratory control ratio (RCR), a key measure of coupling efficiency (V3/V4), was significantly increased by DSIP at 10⁻⁵ and 10⁻⁶ M concentrations, suggesting tighter coupling between respiration and phosphorylation. At 10⁻⁷ M, RCR showed a tendency to increase. Deltaran, an analogue of DSIP, exhibited similar effects, enhancing V3 and ADP/t in brain homogenates, mimicking the action of pure DSIP. The study confirmed that brain homogenates could also be effectively used to evaluate DSIP’s influence on mitochondrial functional state, with DSIP and Deltaran showing comparable activities in both isolated mitochondria and brain homogenates. A crucial observation was that DSIP’s activating effect on oxidative phosphorylation was selective, primarily increasing V3 without affecting VDNP, and showing no evidence of uncoupling oxidative phosphorylation. This suggests that DSIP does not activate all mitochondrial respiration but specifically enhances ATP synthesis [2].

The study then investigated DSIP’s ability to protect against hypoxia-induced mitochondrial dysfunction. In rats subjected to experimental hypoxia, a significant stress-mediated reduction in both the rate of phosphorylated respiration (V3, a 20% decrease) and the rate of ADP phosphorylation (ADP/t, a 31% decrease) was observed in brain mitochondria. This indicates that hypoxia severely impairs the efficiency of mitochondrial energy production. Pre-treatment of rats with 120 µg/kg DSIP 20 minutes prior to subjection to hypoxia completely inhibited these hypoxia-induced reductions in mitochondrial respiratory activity. In other words, DSIP fully protected the rats’ brain mitochondria from the declines in V3 and ADP/t caused by hypoxia, maintaining them at levels comparable to non-hypoxic controls. This demonstrates a robust stress-protective effect of DSIP.

The researchers propose that DSIP’s observed effects, particularly the selective increase in V3 and ADP/t without affecting VDNP, suggest that it does not stimulate the activity of respiratory chain enzymes or substrate transporters, as these would typically affect VDNP. Instead, the increase in V3 and ADP/t is likely attributable to the activation of ATP synthase or adenine nucleotide translocase (ANT). The stability or even slight decrease in V4 (rate of uncoupled respiration) further supports the idea that DSIP may improve the state of the inner mitochondrial membrane by reducing proton leak. Facilitating ANT activity could be linked to DSIP’s membrane-tropic effects and its potential to affect the mitochondrial permeability transition (MPT) pore. DSIP’s known antioxidant properties may also contribute to its influence on ANT, a key component of the MPT pore [2].

The study concludes that DSIP’s activating action on oxidative phosphorylation and its preventive effects under hypoxia provide a strong basis for understanding its previously reported stress-protective and antioxidant actions in vivo. These findings underscore DSIP’s potential as a therapeutic agent for conditions involving mitochondrial dysfunction and oxidative stress, such as cerebral ischemia [1].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Tukhovskaya EA, Ismailova AM, Shaykhutdinova ER, et al. Delta Sleep-Inducing Peptide Recovers Motor Function in SD Rats after Focal Stroke. Molecules. 2021;26(17):5173. Published 2021 Aug 26. doi:10.3390/molecules26175173

[2] Khvatova EM, Samartzev VN, Zagoskin PP, Prudchenko IA, Mikhaleva II. Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia. Peptides. 2003;24(2):307-311. doi:10.1016/s0196-9781(03)00040-8

 

Delta Sleep Inducing Peptide (DSIP) is a nonapeptide that was initially isolated from the cerebral venous blood of rabbits and hypothesized to act as a sleep aid. A study conducted by Stanojlovic et. Al examined the relationship between sleep and epilepsy and whether or not DSIP can play a role in regulating metaphit-induced audiogenic epilepsy.

Metaphit is a synthetically produced substance that was shown to stimulate the subject by increasing brain excitement, and has also been shown to cause seizures in rats. In this study it was administered to 2-month-old male Wistar rats. From there, the rats were exposed to audiogenic stimulation in order to test for any seizure activity or convulsions.

The rats were split into 1 control group and 3 experimental groups. The first experimental group was treated with only 10 mg/kg of metaphit. The second experimental group was treated with metaphit and 1 mg/kg of DSIP. The final experimental group was treated with only 1 mg/kg of DSIP by itself.

The study confirmed that metaphit induced high-voltage activity, and synchronized spike on the EEG only 30 minutes after administration. In the experimental group that received metaphit and DSIP, DSIP was shown to decrease the mean seizure grade as well as the overall duration of the seizure. This indicates that due to its ability to move through the blood-brain barrier, DSIP could be beneficially used against sleep disorders due to the similarity between epilepsy and disturbed sleep (https://pubmed.ncbi.nlm.nih.gov/11797457/).

 

Effects of DSIP on ATP Production

DSIP has been linked to different stress-related metabolic disturbances. As previous studies show, mitochondria are sensitive to stressful conditions, so researchers Khvatova et.Al examined how the administration of DSIP can affect oxidative phosphorylation and ATP production in the brain mitochondria of rats. The initial findings of this study emphasized the increased rate of phosphorylated respiration V3 and ADP phosphorylation as well as the increase of the respiratory control ratio.

The researchers continued on to study the effects of DSIP under hypoxic conditions that alter the respiratory activity in the mitochondria. In rats that were subjected to hypoxic conditions, there was a drastic decrease in V3 and ADP/t levels. However, when the rats were IV injected 120 microg/kg of DSIP prior to being put in hypoxic conditions it was found that the stress-induced decrease in mitochondrial respiratory activity was completely eliminated. Overall this study comes to the conclusion that DSIP could potentially be beneficial in enhancing the efficiency of the oxidative phosphorylation process which could lead to a better understanding of antioxidant and stress-protective behaviors (https://www.sciencedirect.com/science/article/abs/pii/S0196978103000408).

 

Peptides Prefer the Cold
In order to reduce peptide breakdown, keep peptides refrigerated at all times but DO NOT FREEZE.
Swab the top of the vial with 95% alcohol wipe before accessing.
Only Mix with Sterile Bacteriostatic Water
Bacteriostatic water is vital to preventing contamination and preserving the stability of the compound.
Push the needle through the stopper at an angle in order to direct the stream to the side of the vial.
Reconstituted peptide solution should be stored around 4 degrees Celsius but not frozen, while lyophilized peptide solution should be kept at -20 degrees Celsius.

DSIP is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.