1. ClearH 2 O DietGel Treatments After Ovariectomy Bilsky Laboratory University of New England August 1, 2012 Ovariectomy Mouse Eating DietGel 31M

2. Protocol – Ovariectomy Prior to surgery all animals were weighed and baselined in the abdominal von Frey and locomotor assays Mice were anesthetized with isoflurane (2-3%) vaporized via a nose cone Animals were placed in a ventral recumbency with the tail toward the surgeon; the lumbar region of each animal was swabbed with 5% proividone-iodine solution and 70% alcohol A 1-1.5 cm dorsal midline incision was made caudal to the posterior border of the ribs The fascia was cleared away using blunt dissection and the underlying muscle wall was pierced on both sides 1cm lateral to the midline (the ovary was located in a fat pad just beneath the muscles) Using forceps, the periovarian fat was gently grasped to lift and remove the ovary with forceps, no cauterization was needed The uterine horn was returned into the abdomen, and the process was repeated on the other side The muscle incision was not sutured whereas the skin incision was closed using wound clips The sham animals were put under anesthesia for ~4 minutes (length of average ovariectomy) Anesthesia was stopped and animals were allowed to recover before being returned to fresh home cages with either standard food and water, DietGel 76A, DietGel 31M, or DietGel Recovery --------------------------------- Behavioral testing was assessed at 8, 24 and 48 hours post surgery Bodyweights were also recorded 8 hours post surgery as well as every morning at 8am until day 8 post surgery

3. Protocol – von Frey Abdominal von Frey testing was performed on all mice in this study Baseline latencies were taken prior to surgeries and all animals were retested for their response at 8, 24 and 48 hours post surgery Six von Frey filaments each with different gram weights (0.16, 0.4, 1.0, 1.4, 2.0 and 4g) were applied to the abdominal region at each time point with each filament being used 5 times for replication Data was obtained if the animals showed a response to the filament as a “ ✓ ” or if there was no response as a “ ✗ ” A response was considered as any movement or vocalization away from or towards the filament Data was analyzed by determining the % response for each animal to each filament at each time point and the average and SEM for each treatment was graphed

4. Protocol – Locomotor Assay Locomotor activity was assessed with all animals in this study using an activity monitoring system and TruScan software (Coulbourn Instruments, Allentown, PA) Each chamber consisted of 10” x 10” x 16” (l x w x h) arena surrounded by Plexiglas walls The floor was a removable plastic drop pan that was cleaned between sessions 2 sensor rings surrounded the arena on the bottom (tracking total distance) the other positioned 2 in higher (tracking vertical rears) Animal positioning was determined every 100 ms and the software compiled the data into 1 min bins across the total observation time of 30 min Mice were run in this assay prior to surgery, 8, 24, and 48 hours post surgery to determine if the DietGel treatments had any effect on returning locomotion and rearing back up to standard non surgery controls Data was compiled as the total distance (cm) traveled or amount of vertical rears over the course of the 30 min test and group means and SEM’s were graphed

5. DATA: Abdominal von Frey Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 Data shows the effect the ovariectomy has on % response to the filaments over time. Pain hypersensitivity was present at 8 hrs and appears to peak between 24 and 48 hours post surgery.

6. DATA: Abdominal von Frey Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 DietGel 76A The panels above show the effect of DietGels 76A, 31M and Recovery (top to bottom) on relieving the sensitivity caused by the ovariectomy at baseline, 8, 24, and 48 hours post surgery (left to right). DietGel 31M DietGel Recovery

7. DATA: Abdominal von Frey Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 At baseline all testing groups showed no difference in the percent of responses to the filaments At 8 hrs post surgery the pain had not yet fully developed and area under the curve (AUC) analysis was not performed 24 and 48 hrs post surgery showed peak pain and the largest effect of the DietGel treatments with all groups showing a significant difference from the standard food & water group after a non-parametric t-test analysis *, **, *** denotes significance at p < 0.05, 0.01 and 0.001 respectively

8. DATA: Locomotor Assay (Total Distance) Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 A B CD At baseline all groups traveled about 9,000 cm in the 30 min testing period The sham animals had a gradual habituation to the chamber over the course of the experiment traveling an average of about 8,000 cm by the 48 hr time point All ovariectomy groups had a significant decrease in distance traveled vs. the sham animals at the 8 hr time point after a 2-way ANOVA with **, and *** denoting significance at p < 0.01 and 0.001, respectively DietGel 76A showed a positive trend in aiding animals recovery after ovariectomy at 24 and 48 hrs post surgery

9. DATA: Locomotor Assay (Vertical Rears) Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 A B CD The amount of vertical rears was totaled over the course of the 30 min test as the number of times the animal stood on its hind paws and stretched upwards in a vertical manner in the chamber All ovariectomy groups had a significant decrease in vertical rears vs. the sham animals at the 8 hr time point after a 2-way ANOVA with **, and *** denoting significance at p < 0.01 and 0.001, respectively The gels showed no effect on bringing the amount of vertical rears back to sham levels at the 24 and 48 hr time points

10. DATA: Bodyweights Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 Over the course of the experiment bodyweights remained constant in the sham treated group The ovariectomy group receiving standard food & water showed an initial decline in weight after the surgery and then stabilized at day 4 post surgery DietGel 76A showed a significant gain in weight by day 8 post surgery and DietGel 31M was trending in an upward fashion by day 8 DietGel Recovery had a steady increase in weight starting from day 2 post surgery and continued through day 8 All DietGel groups by day 8 had gained weight over the course of the experiment while sham and ovariectomy animals remained around a constant body weight

11. DATA: Difference in Bodyweights from Baseline Researcher: Denise Giuvelis Dates: 5/14-5/16 and 6/5-6/7/12 The graph depicts the difference in bodyweight (Day 8 – baseline) for each group. All three diet gels resulted in greater gains in bodyweight compared to the ovariectomy group placed on the standard food and water regimen.