Golden Retriever Muscular Dystrophy (GRMD)

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Key words: Duchenne muscular dystrophy, Golden retriever muscular dystrophy, Neonatal care, ... (CompuPed, Man's Best Friend Software, Franklin, IL, USA). ..... information in mind, we were motivated to develop a mea- ... Anesthetize and position the dog as described in the ..... Levine, D. (2002) Reliability of goniometry.
Chapter 7 Golden Retriever Muscular Dystrophy (GRMD): Developing and Maintaining a Colony and Physiological Functional Measurements Joe N. Kornegay, Janet R. Bogan, Daniel J. Bogan, Martin K. Childers, and Robert W. Grange Abstract Studies of canine models of Duchenne muscular dystrophy (DMD) provide insight regarding disease pathogenesis and treatment efficacy. To take maximal advantage, colonies of affected dogs must be maintained and outcome parameters developed. In this chapter, we review our 25 years of experience with the golden retriever muscular dystrophy (GRMD) model. Key challenges in colony development (breeding, neonatal death, and the risk of inbreeding) and representative functional measurements (tibiotarsal joint angle and torque force; and eccentric contraction decrement) are discussed. Key words:  Duchenne muscular dystrophy, Golden retriever muscular dystrophy, Neonatal care, Inbreeding, Joint angle, Torque force

1. Introduction Although cellular and gene therapies for Duchenne muscular dystrophy (DMD) are promising, key questions must first be addressed in relevant animal models. Spontaneous forms of X-linked muscular dystrophy due to dystrophin deficiency have been identified in mice (1, 2), multiple dog breeds (3–7), and cats (8, 9). Unlike the dystrophin-deficient mdx mouse, which remains relatively normal clinically, affected dogs develop progressive, fatal disease strikingly similar to the human condition. Accordingly, studies in the canine dystrophin-deficient models, such as golden retriever muscular dystrophy (GRMD) (3, 10), may be more likely than those in mdx mice to predict pathogenesis and outcome of treatment in DMD. Dongsheng Duan (ed.), Muscle Gene Therapy: Methods and Protocols, Methods in Molecular Biology, vol. 709, DOI 10.1007/978-1-61737-982-6_7, © Springer Science+Business Media, LLC 2011

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Most DMD natural history studies have included measurements of muscle strength, joint contractures, and timed function tests. Results from these tests are used to track disease progression and offer insight into clinical milestones, such as the loss of ambulation and the need for ventilatory support. Both muscle weakness and joint contractures contribute to postural instability and ­ultimate loss of ambulation (11). Contracture and muscle strength scores in DMD, for the most part, correlate and deteriorate synchronously over time (12). Contractures generally are attributed to shortening of involved muscles. Otherwise, distal joint contractures in DMD have been attributed to two mechanisms. On the one hand, there may be an imbalance of agonist and antagonist muscles operating around a joint. Weakness of antagonist extensor muscles correlates highly with flexor contracture severity in DMD (12). As opposing extensor muscles weaken, flexor contractures worsen. Alternatively, distal limb flexor contractures in DMD have been attributed to postural instability caused by selective sparing or tightness of proximal flexor muscles (11, 13). Over the past 25 years, we have conducted extensive studies in dogs with GRMD (also termed canine X-linked muscular dystrophy, CXMD). This chapter outlines the strategies and precautions in establishing and maintaining the GRMD colony and functional evaluation of disease progression in GRMD by tibiotarsal joint (hock; ankle) angle and torque force, and eccentric contraction-induced force decline.

2. Materials 2.1. Colony Development and Maintenance

1. GRMD dogs (see Note 1). 2. Reagents used for measuring serum progesterone levels (Biovet–Canada, St-Hyacinthe, Québec, Canada). 3. Reagents used for semen collection (see Note 2): wAg-Tek Semen Collection Cones (Neogen Corp., Lansing, MI, USA), disposable centrifuge tube (sterile, polypropylene, 15  mL) (Thermo Fisher Scientific, Waltham, MA, USA). 4. Reagents used for artificial insemination (AI): Priority Care Nonspermicidal Sterile Lubricating Jelly (5 oz) (First Priority, Inc, Elgin, IL, USA), Pipette 9” w/ flex tip (Veterinary Concepts, Spring Valley, WI, USA), Norm-Ject 10 mL Airtite syringes (Agtech, Inc., Manhattan, KS, USA). 5. Software for calculation of the degree of inbreeding (CompuPed, Man’s Best Friend Software, Franklin, IL, USA). 6. Precision Balance Toploading Scale (Model SB8000, Mettler Toledo, Columbus, OH, USA).

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2.2. Physiological Functional Measurements

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1. Preanesthetic agents: Atropine Sulfate Inj (Baxter Healthcare Corporation, Deerfield, IL, USA), Acepromazine Maleate Inj (Boehringer Ingelheim, St. Joseph, MO, USA), Butorphanol Tartrate (Fort Dodge Animal Health, Fort Dodge, IA, USA). 2. Anesthetic agents: Propofol (Abbott Animal Health, North Chicago, IL, USA), SevoFlo (sevoflurane, Abbott Laboratories). 3. Postanesthetic reversal agent: Naloxone HCl (Hospira, Lake Forest, IL, USA). 4. Pulse oximeter: Cardell Multiparameter Monitor 9405 (Minrad International, Orchard Park, NY, USA). 5. Goniometer (Smith & Nephew Roylan, Germantown, WI, USA). 6. Monopolar Electromyography (EMG) Needle Electrodes (Houston’s Electrode Sales, Penn Valley, CA, USA). 7. TE42 EMG unit (Teca Corp, Pleasantville, NY, USA). 8. Force measuring system with servomotor (Aurora Scientific, Ontario, Canada). 9. Statistical analysis software (Sigma Stat, San Rafael, CA, USA).

3. Methods 3.1. Colony Development and Maintenance

1. For GRMD breeding (see Notes 3 and 4), check for the signs of proestrus in GRMD carriers 3 times a week (Monday, Wednesday, and Friday). At the first sign of proestrus, measure serum luteinizing hormone (LH) and progesterone levels. When LH is positive, ovulation has occurred. 2. Initiate breeding every other day by AI when progesterone levels are >3 ng/mL and vaginal cytology shows >60% cornified epithelial cells. Breeding is continued until progesterone levels are >10 ng/mL and white blood cells are seen on vaginal cytology, indicating the onset of diestrus. 3. Obtain semen from affected male. Uncap the centrifuge tube and place it inside the collection cone. Pull the centrifuge tube down into the cone until about 1 in. of tube is left inside the cone (making a seal with the tube so semen does not leak out). Fold down the top of cone once. Extend the penis from the prepuce. Grasp the bulbocarvernosus muscle of the penis. The penis will engorge and the dog ejaculates into the collection cone. Omit the first and the third fractions. Remove the cone and recap the tube.

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4. Inseminate the bitch by AI. Add a syringe to the flextip part of the pipette. Insert the tip of the pipette in the tube just below the surface of the semen. Draw the sample up slowly without flexing the joint at the end of the pipette. Draw up extra air in the syringe to push the semen through the pipette and avoid leakage. Add nonspermicidal jelly to the tip of the pipette. Place a finger inside the vulva to guide the pipette. Direct the pipette dorsally through the vulva so as to avoid the recess of the vestibule and into the vagina. Attach the syringe to the flex tip of the pipette and infuse the semen. Remove the syringe and add air to force the full volume of semen into the vagina. 5. Evaluate ultrasound and measure relaxin levels 30 days from the onset of diestrus. If confirmed pregnant, the whelping date is calculated to be 27 days later. Body temperature is measured beginning a few days before this date to more precisely predict whelping. With this breeding protocol, conception rates are approximately 85%. 6. Carriers are not removed from the colony at a set age. Rather, we remove them based on the regularity of their estrus cycle, rate of pregnancy, litter size, and pup survival. With these factors in mind, most carriers continue to be bred until they are 5–7 years of age. 7. All bitches are observed at the anticipated time of whelping to ensure that appropriate neonatal care can be provided. 8. Affected pups are weak at birth and require nutritional supplementation for up to 4 weeks. We closely monitor pups during the first 24 h of life to ensure that colostrum is ingested. Colostrum from other dogs is harvested and frozen at −80°C so that it can be used as a supplement for weaker pups that are pushed aside by more vigorous littermates and/or do not have satisfactory weight gain. 9. Pups are weighed soon after birth and, on average, 4 times daily for the first 3 weeks (Table  1). Each pup’s physical response to outside stimuli and hydration level are observed at weighing intervals. Newborn pups are evaluated on their overall responsiveness and strength. Righting response, whereby a pup is placed on its back and allowed to right itself, in addition to rooting, latching onto teats, and suckling ability of the pup when placed near the dam are observed. Any evidence of weakness of these responses or failure to thrive is noted. This would include the aforementioned physical responses and any evidence of respiratory distress, abdominal distension, and/or significant weight loss (i.e., sustained lack of weight gain or loss of 20 g. over a 24-h period). 10. In our hands, survival of GRMD dogs is typically 70–80%. Adequate numbers of dogs can be obtained for long-term

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comparative studies (14–18). An average of 2.25 affected pups from each litter survive beyond 10 days of age. To ­provide an approximate number of affected dogs that can be produced each year by each carrier, the following formula is used: Affected pups/carrier each year = 2.25 (surviving pups/ litter) × 0.83 (estrous cycles/year considering multiple factors) × 0.85 (conception rate) (see Note 4). Using this formula, each carrier produces only about 1.58 affected pups/ annually. Thus, to produce 15 affected dogs each year, one must maintain ten carriers. 11. Based on results of studies to determine the effect of inbreeding on pup mortality (see Note 5 and Table 2), we introduce

Table 1 Schedule for recording dog weights Age group

Frequency to record weight

Neonates (12 months)

1× monthly

Adult affected (>12 months)

1× weekly

Any sternally recumbent dog (any age)

1× daily

Table 2 Mortality rate with respect to % inbreeding Inbreeding (%) 0.00

Group size (n)

Number of litters

Number of pups died

Mortality rate (%)

84

12

4

4.76

4.69–12.89

101

14

13

12.87

15.63–19.53

74

10

14

18.92

20.31–24.22

94

14

21

22.34

25.78–40.63

106

16

34

32.08

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new breeding stock unrelated to the colony, on average, at 5-year intervals. We continue to evaluate this strategy and plan to begin introducing new breeding stock at 3-year intervals. 12. Studies of the effects of various levels of postnatal care on pup mortality have guided methods used to manage pups during the first 2 weeks of life (see Note 6). Pups with partial postnatal care, defined as weighing pups and supplementing those experiencing difficulty with bitch’s milk, milk replacer, or both using gastric tubes or bottles, tend to do better than those that receive no supplement care or are removed from the bitch entirely. 13. Studies of the effect of the GRMD trait on fetal (in utero) mortality have shown that GRMD pups are not at increased risk (see Note 7). 14. Numerous pups have been submitted for necropsy to determine cause of death. Respiratory and intestinal infections are the most common causes of neonatal death. 3.2. Physiological Functional Measurements

Most of our research using GRMD dogs has been focused on preclinical studies of potential treatments for DMD and has been done in collaboration with other scientists. To better utilize the GRMD model in therapeutic trials, we have developed various phenotypic tests to objectively characterize disease progression. Affected dogs have joint contractures (14) and demonstrate weakness of individual (15) and grouped (16) muscles. We have found a correlation between tibiotarsal joint torque and angle measurements (see Note 8). As with mdx mice, weakness is exacerbated by eccentric contractions (17). Importantly, by comparing serial measurements from treated and untreated dogs, one can document improvement or delayed progression of disease (18) (see Note 9). In our hands, results from dystrophin-deficient heterozygous males and homozygous females do not differ (see Note 10). 1. Measurement of the tibiotarsal joint angle. The tibiotarsal joint angle has been used as a surrogate biomarker for contracture severity in GRMD. Initially, we measured this angle in GRMD dogs undergoing serial peroneus longus force measurements (14, 15). Subsequent to this study, so as to be able to compare results with historical values, we have continued to use this basic approach without the need for surgery or immobilization of the limb. Others have described methods to measure the tibiotarsal joint angle at maximal flexion and extension in normal dogs (19, 20). Values for normal dogs using our method approximate, but are somewhat less than those recorded at maximal extension. To measure the tibiotarsal joint angle, first anesthetize the animal using the following agents and doses (also see materials). Preanesthetic agents 20–30 min prior to anesthesia

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induction: atropine sulfate (0.04 mg/kg, IM); acepromazine maleate (0.02  mg/kg, IM) for dogs weighing greater than 5 kg; and butorphanol tartrate (0.4 mg/kg, IM). Anesthetic induction agents: propofol (up to 3 mg/kg, IV – slowly!) for dogs weighing greater than 5 kg; for pups  0.05 indicates a good fit). Mortality correlated positively with inbreeding for the overall colony, including normal, carrier, and affected dogs (Table 2). A total of 375 dogs (81.7%) had some calculated degree of inbreeding, and 81 (21.6%) of these died within the 14-day neonatal period. The coefficient for the indicator variable “affected” was 2.6458. This value must be exponentiated for interpretation. We found e2.6458 = 14.094 (“odds ratio”), implying that the odds of dying, at any level of percent inbreeding, are about 14 times greater for affected pups than for normal/carrier pups. 6. To determine the effect of postnatal care on pup mortality, we have evaluated the effects of care classified as none, partial, or complete. No intervention was defined as monitoring weights only. Partial intervention included weighing pups and supplementing those experiencing difficulty with bitch’s milk, milk replacer, or both using gastric tubes or bottles. Complete intervention involved removing pups experiencing difficulty from the dam entirely and feeding as with the partial group. Litters were grouped according to their level of postnatal care and mortality data were compared for evidence of increased pup survival in any given group. Pups with partial postnatal care tended to do better than those with either no or complete care. 7. We have evaluated the effect of the GRMD trait on in utero mortality by comparing the expected genotypic ratios with the actual ratios of pups born in each litter. An overall evaluation of these data was made in relation to percent inbreeding and mortality rate, as well as an individual comparison by genotype. The Chi-square statistic for each litter was plotted against the percent inbreeding and the plot seemed random with no obvious pattern. Consistent with the plot, the correlation coefficient (specifically Spearman’s Rho) was not significantly different from 0. This suggests that inbreeding does not affect prenatal mortality in our colony. Parity (primiparous or multiparous) also did not affect pup survival. 8. Consistent with the association between unbalanced muscle strength and contractures in DMD (above), tibiotarsal joint extension force and the extension/flexion ratio (Fig. 3a) correlate positively with joint angle in GRMD dogs at 6 months of age. Dogs with weaker extension force have more acute joint angles. Interestingly, flexion values correlate negatively, suggesting that flexor muscle functional hypertrophy can

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b

180 160

Tibiotarsal Joint Angle (degrees)

Tibiotarsal Joint Angle (degrees)

a

140 120 100 80 y = 2.1x + 136 r = 0.62 P < 0.0001 power of test = 0.99 n = 51

60 40 20 0

0

2

4

6

8

10

12

14

16

18

Body-Weight-Corrected Extension Force : Flexion Force Ratio

150

100 y = 182.6 − 10.8x r = − 0.70 P < 0.0001 power of test = 1.00 n = 49

50

0

0

1

2

3

4

5

6

7

Body-Weight-Corrected Cranial Sartorius Circumference (mm/kg)

Fig. 3. Correlations among phenotypic tests in GRMD dogs. Scattergrams with regression lines drawn to show correlations between tibiotarsal joint angle (vertical axis in both) and the body-weight-corrected tibiotarsal extension/flexion force ratio (a) and cranial sartorius circumference (b) (horizontal axis in both) in GRMD dogs at 6 months of age. Joint angle correlates strongly (p