Main Research Findings

1) Dihexa was shown to improve cognitive impairments and recover memory by inhibiting inflammation and decreasing neuronal loss by affecting the PI3K/AKT signaling pathways.

2) Dihexa was found to have the potential to facilitate functional recovery in cases of peripheral nerve injury.

 

Selected Data

1) The research team of Sun et al examined the potential of Dihexa to improve cognitive functioning related to Alzheimer’s disease by targeting the brain AngIV/PI3K/AKT axis. 6 month old male APP/PS1 mice and wild-type C57 mice were used for the purpose of this study. The test subjects were housed in a standard animal room under a 12 hour light/12 hour dark day/night cycle with ad libitum access to food and water. The first part of the study began by randomly dividing the test subjects in four groups including: wild type, APP/PS1, APP/PS1 + Dihexa administered at a dose of 1.44 mg/kg, and APP/PS1 + Dihexa administered at a dose of 2.88 mg/kg [1].

The second part of the study began by randomly dividing the test subjects in three groups including: APP/PS1, Dihexa administered at a dose of 2.88 mg/kg, and 2.88 mg/kg of Dihexa + 0.5 mg/kg of wortmannin. Both Dihexa and wortmannin were prepared by dissolving the compounds in 10% DMSA, 40% PEG 300, 5% Tween 80, and 45% saline. Dihexa was administered to the APP/PS1 mice intraperitoneally from 6 to 9 months of age and 0.9% saline was administered to the wild type group once per day over a three month time period.

After the drugs were administered to the test subjects for 3 months they underwent the Morris water maze test. A round black tub was filled with water and divided into four equal regions labeled as north (N), south (S), east (E), and west (W). A small platform was submerged 1 cm below the water surface, in the center of the northeast quadrant of the tank. Each mouse underwent 4 trials per day for 5 consecutive days. They were allowed 60 seconds to search the tank for the platform and at the end of each session they were placed on the platform and remained there for 30 seconds. On the 6th day the platform was removed and the researchers recorded the number of times the individual mice crossed the quadrant where the platform was previously located, over the course of 60 seconds [1].

After all necessary data was collected for the Morris water maze test, the mice were euthanized and the brain tissue was dissected. The brain tissue obtained was weighed and PBS was added in order to ensure a weight (g) to volume (mL) ratio of 1:9. The samples were homogenized to allow for centrifugation of the supernatant, followed by measurement of AngIV, TNF-alpha, IL-10, and IL-1-beta levels through the use of a sensitive and specific ELISA assay. Following perfusion, the brain was fixed for 48 hours and embedded in paraffin. The 4 um thick paraffin sections were then dehydrated, stained with methylene blue, and washed with distilled water, followed by a repeat of dehydrating and washing the sections in order to prepare for a Nissl staining trial. All positively stained cells were counted and the “% Nissl positive neuron” was calculated by dividing the number of positive neurons by the total number of neurons in each cell [1].

Finally, the samples were prepared for Western blotting analysis by lysing the brain homogenates on ice for 300 minutes in 100 mL of a lysis buffer composed of 120 mM NaCL, 40 mM Tris (pH 8), and 0.1% NP40, followed by centrifugation. Additionally, a bicinchoninic acid (BCA) assay was used to determine the protein concentration; 30 ug of the protein was the separated using 10% SDS-PAGE and electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk and incubated overnight at 4 degrees Celsius, with primary antibodies. This procedure was followed by incubation of the membranes with secondary antibodies at room temperature for 2 hours [1].

2) The research team of Weiss et al examined the potential of Dihexa, mesenchymal stem cells (MSC), and Granulocyte-Colony Stimulating Factor (G-CSF) to promote the recovery of limb damage as a result of nerve damage. For the purpose of this study, 10 to 12 week old inbred male Lewis rats weighing approximately 300 grams each were used. Male rats were used over female rats in order to eliminate any skewed data that may result from hormonal fluctuations related to the rats’ reproductive cycles. All housing, handling, and experimentation that involved the rats were conducted following the procedures set in place by the National Institutes of Health guide for the care and usage of laboratory animals [2].

All of the test subjects included in the study were randomly assigned to 1 of 10 different experimental treatment groups. Group 1 was a vehicle control that received an intravenous injection of saline. Group 2 was a vehicle control that received local Hydrogel and an intravenous injection of saline. Group 3 received local Hydrogel and an intravenous injection of MSC. Group 4 received local MSC via Hydrogel and an intravenous injection of MSC. Group 5 received local MSC and G-CSF via Hydrogel and an intravenous injection of MSC. Group 6 received local MSC and Dihexa via Hydrogel and an intravenous injection of MSC. Group 7 received local MSC and G-CSF via Hydrogel, an intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC and G-CSF dissolved in saline. Group 8 received local MSC and Dihexa via Hydrogel, an intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC dissolved in saline and Dihexa dissolved in DMSO. Group 9 acted as a vehicle control for Dihexa in group 6 and received local MSC and DMSO via hydrogel and an intravenous injection of MSC. Finally, group 10 acted as a vehicle control for Dihexa in group 8 and received local MSC and DMSO via hydrogel, and intravenous injection of MSC, and an injection into the gastrocnemius muscle with MSC and DMSO [2].

The animals underwent a sciatic nerve repair model that began with the transection of the main sciatic nerve above the trifurcation point into the tibial, fibular, and sural branches. Following the nerve transection, heparin was delivered through the tail vein and the animal was monitored under anesthesia for an hour to simulate the transport time it takes to get treatment in cases of traumatic peripheral nerve damage. The surgery was performed on the right hind limb and the left limb served as a control. 1 week after the surgery was performed the rats were administered manual physiotherapy to the right hind limb 1-2 times per week for 5 minutes at a time. Primary outcome measures assessed in this study included limb sensory and motor functioning at various time points until 16 weeks post-surgery. Secondary outcome measures assessed in this included gastrocnemius mass and the presence of a foot-flexion contracture [2].

Sensory functioning was examined using the flexor withdrawal spinal reflex. Testing began 1 week after the sciatic nerve repair surgery was conducted and occurred at weekly intervals. Using forceps, the stimulus was applied to the rats’ hind limb by momentarily pinching the areas innervated by the tibial, fibular, and sural nerves. A normal response to the stimulus was first determined in the unoperated left hind limb and was defined as an immediate withdrawal of the limb with or without a vocalization. The same stimulus was then applied to the operated right hind limb and was graded on a scale of 0-3 in comparison to the left hind limb response. A score of 0 was defined as no response; 1, mild response; 2, moderate response; and 3, normal strong response.

Motor functioning was evaluated starting at 2 weeks post-surgery and testing was conducted on a biweekly basis using a walking track analysis where the rats were made to walk in a confined walkway lined with white paper and led into a dark shelter. The researchers applied water soluble black ink to the plantar surfaces of the rats’ hindpaws prior to walking down the walkway from its entrance and into the shelter. The typical outcome measure used by researchers is the Sciatic Function Index that assesses hind limb motor function based on toe to toe and toe to heel distance. Unfortunately the SFI could not be calculated by the research team as there was poor toe to toe print separation. That being said, an alternative measure was used by the research team that assessed motor function by grading the toe and heel foot print characteristic on a scale from 0-4, with 0 defined as no print, non functional, and 4 defined as complete print, near normal functioning [2].

As it was previously mentioned the secondary outcome measures observed by the researchers included gastrocnemius muscle mass and foot flexion contractures. Foot flexion contracture in the rats were evaluated and graded on a scale of 0-4 with 0 being no contracture; 1, 0-30 degrees; 2, 31-60 degrees; 3, 61-90 degrees; and 4, >90 degrees. Following the end of the sensation and motor studies, the rats were euthanized and both the left and right gastrocnemius muscles were dissected and weighed in order to compare the muscle mass of the operated limb (right) versus the non-operated limb (left) [2].

 

Discussion

1) In order to determine whether AngIV is involved in the development of Alzheimer’s, the research team of Sun et al detected the baseline levels of AngIV in both wild type and APP/PS1 mice. In comparison to the wild type mice, the APP/PS1 mice were found to have significantly lower levels of AngIV in the brain. That being said, Dihexa was administered to the rats in doses of 1.44 mg/kg of 2.88 mg/kg in order to see how levels of AngIV changed in response to the nootropic compound. The results reported that both doses of Dihexa increased levels of AngIV in the brains of APP/PS1 mice, with the 2.88 mg/kg dose increasing these levels to almost the same amount in the wild type. These findings suggest that levels of AngIV in the brain potentially plays a role in the development of Alzheimer’s disease [1].

Figure 1: The average levels of AngIV in the brain in the four different experimental groups included in the first part of the study.

In addition to levels of AngIV in the brain, the test subjects underwent the Morris water maze test in order to measure the cognitive ability of APP/PS1 mice when administered Dihexa. From day 1 of the experiment to day 5 the escape latency was found to remarkably decrease, however, escape latency in the APP/PS1 mice was higher than that of the wild type mice. That being said, both the 1.44 mg/kg and 2.88 mg/kg doses of the nootropic were successful at decreasing the escape latency to various degrees, with this effect being most prominent on the 4th and 5th days of the experiment. Overall, the researchers found that the APP/PS1 mice treated with Dihexa exhibited a significantly better performance in comparison to the control mice when assessing the number of platform crossings. These findings indicate that treatment with Dihexa improves cognition in APP/PS1 mice [1].

Figure 2: Changes in escape latency in the four different experimental groups included in the first part of the study.

Figure 3: The average number of crossing into the target quadrant in each of the four different experimental groups included in the first part of the study.

Following the Morris water maze test, Nissl staining was used to observe the amount of positive neuronal cells present. In comparison to the wild type mice, APP/PS1 mice experienced significant synaptic loss as well as a reduction in the number of neuronal cells in the cerebral cortex. When treated with both the 1.44 mg/kg dose and the 2.88 mg/kg dose of Dihexa, the APP/PS1 mice experienced an increase in the number of neuronal cells present in the cerebral cortex. Based on the results on the Nissl staining, the research team was able to conclude that treatment with Dihexa attenuated the rate of neuronal loss in the brains of APP/PS1 mice [1].

Figure 4: Percentage of Nissl-positive neurons in each of the four different experimental groups included in the first part of the study.

Additionally, in order to explore the mechanism of action being neuronal apoptosis, the research team assessed levels of neuroinflammation and glial activation by detecting levels of IL-1-beta, IL-10, and TNF-alpha in the brain. Baseline measurements found that TNF-alpha and IL-1-beta levels in the APP/PS1 group of mice were much higher than the wild type group of mice. However, when the mice were treated with Dihexa, levels of both IL-1-beta and TNF-alpha were found to significantly decrease. On the other hand, baseline measurements found that levels of IL-10 in APP/PS1 mice were significantly reduced in comparison to wild type mice, and when treated with Dihexa these mice experienced an increase in IL-10 levels. These findings suggest that the nootropic compound elicits neuroprotective effects on nerve cells in the brain damaged by inflammatory factors [1].

Figure 5: Changes in the levels of A) IL-1-beta, B) TNF-alpha, and C) IL-10 in each of the four different experimental groups included in the first part of the study.

It is important to mention that the research team took their experimental procedures a step further in order to define the relationship between Dihexa and the PI3K/AKT signaling pathway, wortmannin, a PI3K inhibitor, was administered to the mice intragastrically to detect the number of neuronal cells and inflammatory factors present. The introduction of wortmannin was found to significantly reverse the expression of PI3K and AKT, as well as the anti-apoptotic and anti-inflammatory effects of Dihexa, resulting in a decrease in the number of neuronal cells present in the cortex and the levels of IL-10, and an increase in the levels of TNF-alpha and IL-1-beta [1].

2) The results of the sensory functioning reported that the fibular nerve boundary was recovered by approximately the first week, followed by recovery of the tibial and sural nerve boundaries. Similar to the fibular nerve the saphenous nerve boundary was recovered earlier, however, the saphenous nerve does not branch off of the sciatic nerve so sciatic nerve transection was not expected to affect the innervation of the saphenous nerve and all response to stimulus of the saphenous nerve was considered normal. Two weeks after the sciatic nerve repair all experimental groups ranged between a 1 and a 2 on the 0-3 graded functioning scale. In groups 7 and 8 including animals receiving injections in the gastrocnemius muscle of G-CSF and Dihexa, the sensory function was more pronounced by week 2 post-surgery. By week 10 sensory function improved in all of the experimental groups with grades on the 0-3 scale ranging from 2.6-3.0. Overall, results of the study found that total sensory function recovery improved early in group 7: mice administered G-CSF; and group 8: mice administered Dihexa [2].

Figure 6: Changes in sensory function grade in experimental groups 1-10 over the course of a 16 week time period following sciatic nerve repair surgery.

In terms of motor functioning, walking track footprints were graded on a scale of 0-4. At two weeks post-surgery all 10 of the experimental groups had grades ranging from 2.3 to 4.00, however, motor function ended up deteriorating by week 16 post-surgery. The exceptions were group 7: mice administered G-CSF; and group 8: mice administered Dihexa, both through injection to the gastrocnemius muscle. These two groups experienced a significant improvement in motor functioning by 16 weeks after the sciatic nerve repair. Additionally, the research team noted that these improvements were seen when the nootropic was administered in the gastrocnemius in combination with mesenchymal stem cells. When administered locally and intraperitoneally there were no remarkable changes in motor functioning observed by the researchers [2].

Figure 7: Recovery of motor functioning in groups of mice administered MSC, G-CSK, and Dihexa, determined by walking track foot prints of normal and sciatic nerve resected limbs.

As for the secondary outcome measures of gastrocnemius muscle mass and flexion foot contracture, there was a significant decrease in muscle mass seen in the right hind limb that underwent sciatic nerve transection, in comparison to the left hind limb that was not operated on. Overall loss of muscle mass ranged from 32-45% and none of the 10 experimental treatment groups experienced any significant changes in muscle mass when administered treatment. Additionally, flexion foot contractures were graded on a scale of 0-4. When compared to the control group, the foot flexion contracture angle was found to be reduced most significantly in group 7: mice administered MSC and G-CSF, with a grade of 1.2; and group 8: mice administered MSC and Dihexa, with a grade of 1.8 [2].

Figure 8: Changes in gastrocnemius muscle atrophy between left and right hind limbs, across the 10 experimental treatment groups.

Figure 9: Foot flexion contracture grade in mice treated with Dihexa and G-CSF, in comparison to the control group of mice.

 

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] Sun X, Deng Y, Fu X, Wang S, Duan R, Zhang Y. AngIV-Analog Dihexa Rescues Cognitive Impairment and Recovers Memory in the APP/PS1 Mouse via the PI3K/AKT Signaling Pathway. Brain Sci. 2021 Nov 11;11(11):1487. doi: 10.3390/brainsci11111487. PMID: 34827486; PMCID: PMC8615599.

[2] Weiss JB, Phillips CJ, Malin EW, Gorantla VS, Harding JW, Salgar SK. Stem cell, Granulocyte-Colony Stimulating Factor and/or Dihexa to promote limb function recovery in a rat sciatic nerve damage-repair model: Experimental animal studies. Ann Med Surg (Lond). 2021 Oct 8;71:102917. doi: 10.1016/j.amsu.2021.102917. PMID: 34703584; PMCID: PMC8524106.

 

 

 

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