Effects of Polyhydroxyfullerenes on Organophosphate-Induced Toxicity in Mice

Abstract

Two polyhydroxyfullerenes, which decrease organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition in vitro, were administered by the intraperitoneal (ip) route or applied topically at doses of 0.9–24 mg/kg to protect adult male mice from enzyme-inhibiting and behavioral effects indicative of OP toxicity resulting from exposure to 1.7 – 2 mg/kg diphosphorofluoridate (DFP) ip or 2.3 – 2.7 mg paraoxon topical. Dosing paradigms included OP-fullerene simultaneous administration by the ip route, and 20 min post-OP polyhydroxyfullerene treatment topically. Benefits of OP sequestration by the polyhydroxyfullerene were noted and were dependent on the OP compound as well as timing and route of the polyhydroxyfullerene treatment.

1. Introduction:

Organophosphate (OP)-induced toxicities occur as a result of overexposure to certain insecticides and pose a threat when nerve agents are used in acts of terror (Costa, 2019). Their primary mode of acute toxicity depends on the capability of these compounds to irreversibly inhibit acetylcholinesterase (AChE) activity of the nervous system, leading to cholinergic poisoning. Clinical evidence of poisoning occurs when AChE activity is rapidly and dramatically reduced. This emergency can occur within minutes of exposure, as OP compounds are lipophilic substances that are rapidly absorbed by oral, dermal and inhalation routes. Standard treatment of cholinergic poisoning includes parenteral administration of high doses of the anticholinergic drug atropine, the enzyme regenerator pyridoxime (2-PAM), and benzodiazepine anticonvulsants if seizures occur. Antidotal doses of atropine alone, which are 3 to 10-fold higher than usual pre-anesthetic doses, can counteract OP poisoning if seizures do not occur and 2-PAM is unavailable. However, atropine itself can be toxic so it is inappropriate to use high doses of this agent unless serious or life-threatening symptoms of poisoning are present (Drug Facts and Comparisons, 2017; Suchard, 2015). None of the treatment agents given by routes that cause systemic effects (e.g., intravenous, intramuscular) are useful for prevention of poisoning.

Prevention of OP poisoning has value for protection of military personnel when exposures to nerve agents are probable, and include pretreatment with the reversible cholinesterase inhibitor pyridostigmine, which was used in the 1991 Gulf War (Institute of Medicine, 2000). This drug, however, has deleterious effects of its own and can worsen poisoning if used after exposure has occurred. When exposure to OP nerve agents is dermal, post-exposure application of an available topical nucleophilic scavenger (RSDL®, Emergent Biosolutions, Gaithersburg, MD) can be effectively used, but this substance needs to be quickly removed as it, too, can have deleterious effects (Medical Countermeasures Defense, 2020; Emergent Biosolutions, 2020; Taysse et al., 2007).

Because the current treatments and preventative approaches for OP poisoning can result in toxicities, there currently are no products that can be used safely by the general public for prophylaxis during threats of mass exposures, including substances that could be administered or applied after exposure but before onset of signs of toxicity. In the experiments described herein, we suggest that polyhydroxyfullerenes that have capability to sequester OP compounds (Magnin et al., 2019) could fit these criteria.

Polyhydroxyfullerenes, as used in the present experiments, are water-soluble spherical structures containing 60–80 carbons with 14–20 hydroxyl groups attached (Ehrich et al., 2011). In this study they were investigated for their potential to provide a safe intervention that could be applied at or after exposure to OP toxicants. These non-cytotoxic substances decreased OP-induced AChE inhibition in vitro (Ehrich et al., 2011). When administered to rodents by intravenous (IV) or intraperitoneal (ip) routes, they did not cause clinically significant effects on clinical chemistry nor did their administration result in treatment-associated histopathology (Jortner et al., 2012; Monteiro-Riviere et al., 2012; Wallace et al., 2014).

2. Materials and Methods:

2.1. Test chemicals.

Synthesis and characterization of polyhydroxyfullerene test agents were performed by Luna NanoWorks, Danville, VA, as described previously (Ehrich et al., 2011). Those products provided in limited quantities by Luna and used for the present experiments were a gadolinium-containing 80 carbon proprietary trimethosphere (Gd₃N@C₈₀-OH; Gd-TMS) and a fullerene with 70 carbons derivatized and solubilized with tetraglycolic acid (C70-TGA). Purity was evaluated by reverse phase TLC (Gd-TMS) or by HPLC (C70-TGA) as >95%. The test organophosphorus (OP) compounds were diisopropylphosphorofluoridate (DFP; Sigma-Aldrich, St. Louis MO, lots 108K144 and 0099K1346, >99% pure) and paraoxon (Lot 407–31B, Chem Services, West Chester, PA, >99% pure). Ethanol USP 100% was from Decon Labs, King of Prussia, PA.

2.2. Animal dosing.

Adult outbred white male mice (ICR (CD-1®)), 28–31 gm, used for these experiments were obtained from the Dublin, VA, facility of Harlan Sprague Dawley (now Envigo). They were housed 5 to a cage on Diamond Dry Cellulose Bedding (Harlan-Envigo) in the laboratory animal facility at the College of Veterinary Medicine where they were provided with standard pelleted rodent chow and water ad libitum. The facility operates on a 12 h light-dark cycle. The mice were randomly assigned to treatment groups. Two different dosing routes were used for these experiments, intraperitoneal (ip) and topical. Preliminary experiments were done to select OP doses that caused notable clinical deficits without need for atropine rescue to assure survival. Dosing and behavioral observations were done by different laboratory personnel. Polyhydroxyfullerene doses were selected based on tolerated doses for their use as MRI contrast agents (Gd-TMS) or anti-inflammatory agents (C70-TGA) and previous safety studies performed in our laboratory and others (Adiseshaiah et al., 2013; Dellinger et al., 2015; Jortner et al., 2012; Monteiro-Riviere et al., 2012; Murphy et al., 2016; Wallace et al., 2014). None of the polyhydroxyfullerene doses chosen caused any detrimental effects in these studies and other previous experiments. All protocols received prior approval from Virginia Tech’s Institutional Animal Care and Use Committee (IACUC).

For experiments evaluating systemic administration of a polyhydroxyfullerene on OP toxicity, the OP test compounds and Gd-TMS were administered together using the ip route. In this case, the OP and Gd-TMS dosing solutions were prepared in 2% ethanol, a vehicle compatible with the polyhydroxyfullerene powder and the lipophilic OP product. The OP dosing solution was first prepared in cold 100% ethanol and then diluted 50-fold in saline to make the dosing solution immediately before its administration. The control group received 2% ethanol vehicle only. DFP doses were 1.7 and 2 mg/kg and Gd-TMS doses were 8 and 24 mg/kg. The products were administered so the mg/kg ip doses were in 5 ml volumes. As noted above, OP doses were chosen to provide time to observe clinical deficits, deficits that were evident without needing rescue with atropine before sacrifice 30 min after dosing.

For topical exposures, paraoxon was prepared in cold 50% ethanol with the 2.3 – 2.7 mg/kg dosage applied to dorsal skin in a 10 μL volume. (Paraoxon could provide reproducible time of response; DFP was not used after preliminary experiments demonstrated too wide a variability in time of onset and magnitude of signs.) For these experiments, the mouse dorsum was shaved the day before dosing. Polyhydroxyfullerene in 50% ethanol was prepared as a paste so the dosage applied was in 4 × 10 μL volumes of 1 – 1.6 mg/ml suspensions with application 20 minutes after paraoxon dosing.

Assessments of polyhydroxyfullerene effectiveness included time of onset of clinical signs, number showing clinical signs of cholinergic toxicity, and behavioral effects using a mouse functional observational battery that evaluates general activity, posture, mobility, gait and presence/absence of involuntary movements (Table 1). Brain cholinesterase activity was evaluated after sacrifice by pentobarbital overdose at 30 minutes (ip) or 80 min (topical) after OP dosing. This enzyme activity in phosphate buffer-rinsed mouse brain was determined using suspensions of whole brain homogenates. The spectrophotometric microtiter assay measured absorbance at 412 nm of the enzymatic breakdown of acetylthiocholine substrate (Correll and Ehrich, 1991; Ellman et al., 1961). Control values ranged from 7–11 μmoles acetylthiocholine hydrolyzed per minute per gram of tissue.

Table 1:

Neurobehavioral Assessment Endpoints.

OBSERVATIONS (example after paraoxon topical 2.2 mg/kg)		Time after OP Dosing
Test Name:	Score*	0 min	10 min	20 min	30 min	40 min	50 min	60 min	70 min	80 min
Posture	1–7	1	1	1	4	6	6	6	6	6
Activity	1–7	5	5	5	3	3	3	3	3	3
Gait	1–9	1	1	1	1	7	7	7	7	7
Gait Score	1–4	1	1	1	1	2	2	3	3	4
Mobility Score	1–4	1	1	1	1	1	2	3	3	3
Clonic - Involuntary motor movements	1–10	1	1	1	1	4	4	5	5	5
Straub tail	Y/N	N	N	N	N	N	N	N	N	N
Salivation	1–3	1	1	1	1	2	2	2	1	1
Lacrimation	1–3	1	1	1	1	1	1	1	1	1
Ocular Discharge	Y/N	N	N	N	N	N	N	N	N	N
Respiration	1–2	1	1	1	1	1	1	2	1	1
Piloerection	Y/N	N	N	N	N	N	N	N	Y	Y
Diarrhea/Soft Stool	YD,YS/N ND	ND	ND	ND	ND	ND	ND	ND	ND	ND

^*Categorical rating; Y/N = appeared or not; YD = presence of diarrhea, YS = presence of soft stool; N = no diarrhea; ND = no feces. Description of categories is provided in Appendix A.

2.3. Statistics.

AChE activities (expressed as percent of control) were summarized as mean ± SEM and compared between groups using analysis of variance followed by Tukey’s procedure for multiple comparisons. Neurobehavioral Assessment scores (Table 1) were compared between groups using Fisher’s Exact test followed by Bonferroni’s procedure for multiple comparisons. Time to first abnormality was compared between groups using the Wilcoxon rank sum test followed by Bonferroni’s procedure for multiple comparisons. Statistical significance was set to p< 0.05. Data were analyzed using SAS version 9.4 (Cary, NC, USA).

3. RESULTS

3.1. DFP ip and ip Gd-TMS.

For these studies, ip DFP was used as the test OP compound, because it provided a relatively longer onset and more moderate dose-response for evaluation of toxicity using behavioral endpoints than another potent, volatile OP compound with a steep dose-response curve, paraoxon. Effects of DFP were dose-related, and mice could survive the 30-min test period without requirement for atropine rescue. These in vivo studies suggested that ip gadolinium trimetasphere (Gd₃N@C₈₀-OH; Gd-TMS) at 8 mg/kg or 24 mg/kg had potential to protect mice from toxic but non-lethal ip doses of DFP when given simultaneously. Results were reproducible among 3 different experiments in which DFP was dosed at 2 and at 1.7 mg/kg. Mean AChE activities were higher in DFP-treated mice given Gd-TMS than in mice given only DFP when evaluated at 30 min (Figure 1). Improvement in brain AChE activity in mice given Gd-TMS with 2 mg/kg DFP averaged 10% over AChE acitivity in mice given only DFP. Beneficial effect of the Gd-TMS on brain AChE activity was more notable with 1.7 mg/kg DFP, as mean AChE activity was >20% higher in brains of 13/18 (72%) Gd-TMS-treated mice and AChE activity differences between DFP-treated and DFP + Gd-TMS brain homogenates were statistically significant (Figure 1; p < 0.05). Diaphragm and blood samples were also collected and analyzed for cholinesterase activities. Results were more variable but showed similar trends as brain AChE activities (data not shown).

Figure 1: Brain AChE activities in mice given intraperitoneal DFP with and without the polydroxyfullerene Gd₃N@C₈₀-OH (Gd-TMS).

Results were obtained following sacrifice 30 min after dosing. Values from individual mice as well as the group mean are provided. The beneficial effect of Gd-TMS on AChE inhibited by 1.7 mg/kg DFP was significant (p<0.05), n=6–9.

Clinical scores were obtained on groups of mice that provided brain samples used for the AChE data provided in the Figure 1. Comparative results are presented in Table 2. The mice treated with 2 or 1.7 mg/kg DFP showed definite clinical differences from vehicle-treated controls. Considering DFP-induced effects on general activity, mobility score, gait description, gait score, involuntary movements, and posture, differences were p<0.05 for 4 of these indices as early as 5 min after dosing and at p<0.05 for all 6 of the listed indices 15–30 min after dosing. The Gd-TMS-treated mice showed greater benefit against 2 mg/kg DFP-induced detrimental effects than when given 1.7 mg/kg DFP, probably because clinical signs with this dose of DFP alone were more dramatic. With this higher dose of DFP, significant improvements (p<0.05) were seen in general activity, mobility, gait and involuntary movements. When the DFP dose was 1.7 mg/kg, most notable improvements were on gait.

Table 2: Clinical score comparisons measured during the 30 min observation period after DFP administration with and without Gd₃N@C₈₀-OH (Gd-TMS).

Clinical scores were statistically compared between the DFP- and DFP plus Gd-TMS at the earliest time interval observed between 5 and 30 min after DFP dosing. Once observed, the clinical sign for an individual mouse did not change over the 30-minute test period.

Measurement	DFP-dosed compared to control (n = 9)	DFP 2 mg/kg. Comparison of DFP alone and DFP + Gd-TMS (n = 10–16)	DFP 1.7 mg/kg. Comparison of DFP alone and DFP + Gd-TMS (n = 8–12)
General Activitya	p < 0.01	p = 0.018	p = 0.078
Postureb	p < 0.01	p = 0.109	p = 0.18
Mobility Scorea	p < 0.03	p = 0.046	p = 0.39
Gait Descriptionc	p < 0.001	p = 0.051	p = 0.042
Gait Scorec	p < 0.001	p = 0.037	p = 0.20
Involuntary Movementsb	p < 0.01	p = 0.046	p = 0.12

Time of onset of signs used for the statistical comparison between DFP alone and DFP + Gd-TMS groups indicated by superscripts:

^a5–15 min

^b5–20 min

^c10–15 min; n = 9 each for vehicle-treated mice, mice given DFP 2 mg/kg alone and mice given DFP 1.7 mg/kg alone.

Additional experiments with ip DFP (n=6–9) examined the possibility that Gd-TMS could be given 3 or 15 minutes after administration of the OP. These did not provide strong indication of significant polyhydroxyfullerene benefit against DFP-induced effects, although modest improvements of general activity (p = 0.083), posture (p = 0.054) and gait (p = 0.028) were seen at observation times 15–20 min after 2 mg/kg DFP when Gd-TMS was given after the OP. As DFP toxicity was evident in ≤ 5 min after ip injections, providing such a narrow window for effective treatment, further benefits of post-exposure systemic administration of polyhydroxyfullerenes in OP toxicities were not explored.

3.2. Topical administration of paraoxon and 20 min post-application of topical polyhydroxyfullerenes.

The topical route of OP administration was explored as it provides a slower rate of absorption than the ip route used for the experiments described above with DFP. Topical administration also has the advantage of providing a real-world relevant route of OP exposure and a safe and useful route for application of a countermeasure. Preliminary experiments indicated that DFP would not be a good OP for these experiments because time of onset and severity of signs were exceedingly variable. However, we found that paraoxon applied topically in a 10 μL volume of 50% ethanol at a dose of 2.3–2.7 mg/kg would consistently lead to clinical evidence of non-lethal OP toxicity with an onset of approximately 20 minutes without lethality or need for atropine rescue for as long as 80 minutes after dosing. This dosing paradigm was used for time-related evaluation of clinical signs between OP dosing and sacrifice 80 minutes later. Differences between paraoxon and vehicle treatments were statistically significant on all behavioral parameters evaluated (Table 3).

Table 3: Effects of topical paraoxon on mouse behavior and benefit of treatment with polyhydroxyfullerenes, Gd-TMS and C70-TGA, n= 6–9.

Gd-TMS and C70-TGA were administered 20 min after paraoxon (Px). Comparisons of polyhydroxyfullerene benefit were made by comparing behavioral indices (Table 1, Appendix A) between the paraoxon + polyhydroxyfullerene-treated group with the group of mice given only Px.

MEASUREMENT	Paraoxon (Px) effect, compared to vehicle	Px + GdTMS topical compared to Px alone	Px + C70-TGA topical compared to Px alone	Px + GdTMS ip compared to Px alone	Px + C70-TGA ip compared to Px alone
Mobility (Mobl)	p < 0.04 at ≥ 40 min	p = 0.12 at 80 min; onset time p = 0.128	p = 0.068 at 70 min	No difference (p>0.25)	No difference
Gait description	p < 0.04 at ≥ 40 min	p = 0.13 at 60 min; onset time p = 0.17	p = 0.045 at 60 min	No difference	No difference
Gait score (GS)	p < 0.04 at ≥ 40 min	p > 0.2	p = 0.09 at 70 min; onset time p = 0.17	No difference	No difference
Involuntary movements	p < 0.04 at ≥ 50 min	p = 0.2 at 60 min	p = 0.22 at 70 min	p = 0.14	p > 0.2
General Activity (GA) score differences	p < 0.02 at ≥ 20 min	p > 0.15	p > 0.15	p = 0.06 at 40 min; onset time p = 0.005	p = 0.05 at 40 min; onset time p = 0.07
Posture (Pos)	p < 0.006 at ≥ 20 min	p = 0.24 at 70 min	p = 0.15 at 70 min	p = 0.006 at 30 min; onset time p = 0.017	p = 0.11 at 50 min; p = 0.031 at 60 min; onset time p = 0.12
Piloerection	p < 0.09 at ≥ 50 min	p > 0.2	p = 0.07 at 50 min	p = 0.005 at 50 min	p = 0.01 at 50 min

Two solubilized polyhydroxyfullerenes, Gd-TMS and C70-TGA, were given either by the ip route (8 mg/kg for GD-TMS; 0.9 mg/kg for C70-TGA) or applied topically (3.7 mg/kg for Gd-TMS; 0.9 mg/kg for C70-TGA). The ip doses were prepared in saline and provided in volumes of 0.1 ml. Topical application was 4 × 10 μL spots as a 50% ethanol paste. Dosing was 20 min after paraoxon, and observations were made every 10 minutes until sacrifice 80 minutes after OP dosing. Results presented in Table 3 note the earliest times in the 80 min observation period when polyhydroxyfullerene-treated mice were notably different (p < 0.1) from paraoxon-only treated mice. Intraperitoneal polyhydroxyfullerenes were better than topical products on some endpoints evaluated. Application of the 50% ethanol vehicle had no effect on behavior and did not alter effects of paraoxon or the polyhydroxyfullerenes.

The behavioral indices demonstrated improvement in 4/7 of the endpoints evaluated when C70-TGA was given topically 20 minutes after administration of topical paraoxon (Table 3). Statistical improvements (p<0.05) were noted with 3/7 endpoints (general activity, posture, piloerection) when ip Gd-TMS was administered 20 min after topical paraoxon (Table 3).

Time until onset of signs is noted in Figure 2. Topical paraoxon (Px) caused clinical signs to appear in mice ≅ 20 min after exposure. Once they appeared, they continued throughout the time of observation. Figure 2 shows that topical administration of solubilized fullerenes delayed onset of paraoxon-induced clinical signs.

Figure 2: Effects of topically applied polyhydroxyfullerenes Gd-TMS and C70-TGA on mean time of onset of clinical signs induced by topical paraoxon, n=6–9.

Fig 2a = mobility; Fig 2b = gait alterations; Fig 2c = involuntary movements. The lines on the figure represent the time to onset of change from vehicle-treated mice in paraoxon- and paraoxon + polyhydroxyfullerene-treated mice, expressed as a % of the total mice dosed.

AChE activities were measured in mouse brains collected 80 min after paraoxon (Px) administration. Mean enzyme activities were higher in all paraoxon mice treated with Gd-TMS or C70-TGA than in mice administered only paraoxon. Polyhydroxyfullerenes protected some, but not all, mice from paraoxon-induced AChE inhibition. For the data presented in Figure 3, 25% of the OP + polyhydroxyfullerene-treated mice had AChE activities ≥ 20% of paraoxon median. An overall analysis of variance provided F_4,50=0.62, p=0.654.

Figure 3: Paraoxon effects on brain AChE activity in the absence and presence of polyhydroxyfullerenes Gd-TMS and C70-TGA.

Polyhydroxyfullerene administration protected some, but not all, mice from paraoxon-induced AChE inhibition. Results are presented as mean ± SEM of n=6–9. An overall analysis of variance provided F_4,50=0.62, p=0.654.

4. DISCUSSION

The results presented indicated that polyhydroxyfullerenes could provide some protection against early acute OP toxicity. The results demonstrated an effectiveness that would have to be supported by use of other countermeasures to assure complete protection, especially if victims were exposed to lethal doses of OP insecticides or nerve agents. However, delay in onset and less severe signs would better allow triage and control in a situation of mass poisonings, such as occurred in the Tokyo subway (Okumura et al., 1996). The chemical nerve agent used there (sarin) has very rapid absorption, a very steep dose-response curve and a very short time to onset of symptoms (Institute of Medicine, 2000). Exposure to paraoxon and DFP at the doses used in the present studies showed rapid and notable effects not dissimilar to sarin.

Unlike lipophilic OP compounds that are quickly absorbed systemically after dermal exposure, the polyhydroxyfullerenes used in the present experiments can be dissolved in water and would be unlikely to be systemically absorbed after dermal application. This would diminish the possibility of any detrimental systemic effects. That they would not be dermally absorbed also suggests the possibility of safe prophylactic use after topical OP exposure. Even when given by routes resulting in systemic exposure (e.g., ip, IV, oral), the polyhydroxyfullerenes used in these studies and similar compounds used in other studies did not cause toxicity even at doses higher than the 4 mg/kg of topical product applied in these experiments (Jortner et al., 2012; Monteiro-Riviere et al., 2012; Quick et al., 2008; Wallace et al., 2014). The topical use of these polyhydroxyfullerenes provides an element of safety not noted with RSDL®, a skin decontaminant that is not to be applied unless exposures occur and is to be washed off as soon as possible afterwards (Medical Countermeasures Defense, 2020).

That non-toxic polyhydroxyfullerenes could be applied topically and delay onset of signs of cholinergic poisoning in the experiments presented here suggest that they would have value in situations of mass dermal poisonings. One consideration is that they could be applied by non-medical personnel to victims that, unknown at time of exposure, may not be poisoned enough to ever show cholinergic signs. Furthermore, they could be applied to possible victims in the time between exposure and appearance of symptoms of poisoning. If such application to potential victims resulted in even a 20 minute delay until the onset of signs of toxicity, this could provide emergency medical personnel with critical time to provide support to seriously ill patients (Jett, 2016).

Although fullerenes could protect AChE from inhibition in vitro (Ehrich et al., 2011), the polyhydroxyfullerenes used in these experiments could not provide complete protection from OP poisoning. This likely has to do with how they interact with OP compounds. Polyhydroxyfullerenes exist as nanoscale aggregates in aqueous solution where they form stable nanoparticles. Rather than inactivating the OP compound, polyhydroxyfullerenes sequester them in a manner similar to cyclodextrin (Magnin et al., 2019). Therefore, their action, when applied topically, would be to delay or decrease the dermal absorption of the OP poison. With a longer delay in absorption, the peak systemic concentration would be lower, with possibility of a decrease in severity of poisoning. The intensity of binding of OP compounds to polyhydroxyfullerenes, however, is considerably less than binding to AChE (Magnin et al., 2019), so polyhydroxyfullerenes could not be relied upon to be the sole treatment agent in cases of severe poisoning. However, use prophylactically or as decontamination products immediately after OP exposure has potential to be of benefit when a large number of victims have both temporal and severity differences in poisoning, as occurred in the Tokyo subway (Okumura et al., 1996). How the hydroxyfullerenes would compare in efficacy, economy, and ease of use to other proposed topical agents reported to have potential value for OP decontamination before or after dermal OP exposure (e.g., nucleophiles, hemostatic decontaminants, fuller’s earth, magnesium sulfate solutions, oxime-chitosam-containing products) (Dachir et al., 2017; Lydon et al., 2017; Taysse et al., 2007; Thorat et al., 2018; Wong et al., 2019) remains to be determined.

Appendix A

Description of Behavioral Categories

Posture

1. Sitting or standing normally (normal).

2. Rearing (normal).

3. Lying on side or curled up (normal).

4. Lying flat, limbs may be spread out.

5. Lying on side.

6. Crouched over.

7. Head bobbing.

Activity Level

1. Stupor, coma.

2. Sleeping.

3. No exploratory movements.

4. Minimum exploratory movements.

5. Normal (alert, exploratory movements).

6. Somewhat high (slight excitement, sudden darting or freezing).

7. Very high (more than 6).

Gait - If mouse did not move during the observation period, it may be gently prodded in order to observe the gait. Scoring is as follows. More than 1 score can be recorded if necessary. The most prominent abnormality is entered under “Gait” on Raw Data Sheet. Less prominent changes are listed on back under “Observation Comments”.

1. Normal.

2. Ataxia, excessive sway, rocks or lurches.

3. Hind limbs show exaggerated or overcompensated movements, drag, or are splayed.

4. Feet markedly point outward from body.

5. Forelimbs drag, unable to support weight.

6. Walks on tiptoes.

7. Hunched or crouched body position.

8. Body drags or is flattened against surface.

9. Lame (describe under other).

Gait score - Rank the overall degree of gait abnormalities, and score as indicated below.

1. Normal, i.e., no abnormal gait.

2. Slightly abnormal.

3. Moderately abnormal.

4. Severely abnormal.

Mobility score - Rank ability of mouse to locomote despite gait abnormalities.

1. Normal.

2. Slightly impaired.

3. Moderately impaired.

4. Totally impaired.

Clonic - Involuntary motor movements - Colonic movement can be defined as rapid contraction and relaxation of muscles.

1. None.

2. Repetitive movements of mouth and jaw.

3. Quivers of limbs, ears, head, or skin (normal).

4. Mild tremors – site specific (e.g., only in tail, abdomen, hind limbs, etc.)

5. Mild tremors - whole body.

6. Severe whole body tremors.

7. Myoclonic jerks.

8. Clonic convulsions.

9. Wet dog shakes.

10. Severe clonic convulsions resulting in dyspnea (breathing difficulty), postictal depression, or death.

Straub Tail - Y indicates the presence of Straub tail. N indicates normal tail posture (no Straub tail present).

Salivation - Observe salivation (drooling)

1. None.

2. Slight.

3. Severe.

Lacrimation - Observe tearing and crusty secretions around eyes

1. None.

2. Slight.

3. Severe.

Ocular Discharge - indicated as the presence (Y) or absence (N) of dark material around eye(s). Respirations

1. Normal.

2. Abnormal (labored, increased rate, etc.).

Piloerection - Observe hair standing on end. Can be verified (and differentiated from simply lack of grooming) by running a hand over hair and see whether or not it returns to the erected state. Y indicates presence of piloerection (i.e., coat does not lie down after stroking). N = no piloerection Diarrhea/Soft Stool - indicated as present (YD = presence of diarrhea, or YS = presence of soft stool) or absent (N). If no feces, use ND for not determined.