Survival, growth curves and general appearance
Newborn pups (n
=82) from 10 litters of SMNΔ7 carrier (SMN2+/+;SmnΔ7+/+;mSmn+/−
) matings were analyzed for survival as well as changes in motor behavior. Of these pups, 24 had SMA (SMN2+/+;SmnΔ7+/+;mSmn−/−
), 35 were carriers and 23 were normal (SMN2+/+;SmnΔ7+/+;mSmn+/+
). Within this testing group, the average lifespan of SMNΔ7 SMA mice was 13.6 ± 0.7 days, which is in close agreement with previous results (Le et al., 2005
). Male SMNΔ7 SMA mice tended to live longer on average than their female littermates (15.0 ± 0.6 days versus 12.5 ± 1.2 days), however, this trend was not statistically significant (; χ2
=0.834, p=0.36). There is no correlation between the mass of SMNΔ7 SMA mice at birth and their lifespans (Pearson R = 0.054). The size of the litter has no correlation with lifespans of SMNΔ7 SMA mice (Pearson R = −0.026). Additionally, maternal age has no effect on the survival of SMNΔ7 SMA mice (Pearson R = 0.176).
Survival curves of male and female SMNΔ7 SMA mice
During the early stages of the disease—i.e. PND02-PND07, SMNΔ7 SMA mice are not significantly different in appearance aside from their smaller size when compared to their carrier and normal (i.e., non-SMA) littermates (). In fact, the appearance of body fur and the eruption of incisors occur in SMNΔ7 SMA mice at the same times as for non-SMA mice. SMNΔ7 SMA mice are smaller and appear emaciated at PND11 and PND14 () presumably due to neurogenic muscle atrophy. Postnatal growth curves were analyzed for SMNΔ7 SMA, carrier and normal mice of both sexes. Both male and female carriers as well as normal pups have a continuous growth curve (). The growth curves for both male and female SMNΔ7 SMA mice are different from their non-SMA littermates (testing day F13,858 = 1263, p < 0.001; testing day×genotype F65,858 = 43.2, p = 0.002). For male pups (), the mean body masses between SMNΔ7 SMA and normal mice are significant different beginning at PND03; however, the differences in mean body mass between SMNΔ7 SMA and carrier mice are significant starting at PND05. There is a significant difference in body mass between female SMNΔ7 SMA mice and their normal littermates beginning at PND02 while body masses of female SMNΔ7 SMA mice are significantly different from carrier mice starting at PND03 (). Male and female SMNΔ7 SMA mice continue to gain body mass until PND09-PND11 where after they start losing body mass. The mean onset of body mass loss () occurs later in male SMNΔ7 SMA mice (11.1 ± 0.3 days) than in female SMNΔ7 SMA mice (9.8 ± 0.7 days after birth) but, as with survival, this difference is not significant (χ2 = 0.252, p=0.62).
General appearance of SMNΔ7 SMA mice
Body mass growth curves of SMNΔ7 SMA mice
Onset of Reflexological Responses
Neonatal mice will normally reposition themselves to a prone position within 5 seconds after being placed on their backs (righting reflex response). The righting reflex response can be seen in pups as early as PND02. However, only 10–30% of SMNΔ7 SMA are able to exhibit a righting reflex response at any given age (; Mann-Whitney U = 170.5, p < 0.001 when comparing SMA to carrier mice at PND02; U = 87.5, p < 0.001 at PND03; U = 61.5, p < 0.001 at PND04; U = 45.0, p < 0.001 at PND05; U = 43.0, p < 0.001 at PND06; U = 32.0, p < 0.001 at PND07; U = 64.0, p < 0.001 at PND08). Of the 24 SMA mice tested, only 7 were able to right themselves for two consecutive days whereas all of the carrier and normal mice were able to consecutively right themselves. Likewise, the amount of time required for a mouse to reposition itself from a supine position (righting reflex latency) is about 2.5–5-fold greater for SMNΔ7 SMA mice than for their non-SMA littermates (; testing day F6,444 = 1.193, p < 0.001; testing day × genotype F6,444 = 3.605, p = 0.002). The sex of the mouse had no effect on the righting reflex response nor on its latency.
Acquisition of reflex responses in SMNΔ7 SMA mice
As shown in , only 15–30% of SMNΔ7 SMA mice were able to elicit a proper response to negative geotaxis (Mann-Whitney U = 212, p < 0.001 when comparing SMA to carrier mice at PND04; U = 124, p < 0.001 at PND05; U = 84, p < 0.001 at PND06; U = 100., p < 0.001 at PND07; U = 92, p < 0.001 at PND08); most non-SMA mice, on the other hand, demonstrate a negative geotaxis response as early as PND04. There is no difference in the proportion of carrier and normal mice who exhibited a negative geotaxis response (e.g., Mann-Whitney U = 321.5, p = 0.313 at PND04). The latency to a negative geotaxis response is significantly longer for SMNΔ7 SMA mice (; testing day F4,296 = 2.47, p = 0.045; testing day × genotype F4,296 = 1.50, p = 0.203) than for non-SMA mice.
When placed over a ledge, neonatal mice will normally turn and move away from ledge (cliff aversion). Fewer SMNΔ7 SMA mice were able to successfully demonstrate a cliff aversion response () than non-SMA mice at PND06 (17.4%; Mann-Whitney U = 179.0, p < 0.001 when comparing SMA to carrier mice), PND07 (28.6%; U = 169.5, p < 0.001) and PND08 (33.3%; U = 112.0, p < 0.001).
Onset of Sensory Responses
The onset of tactile sensory functions was assessed in SMNΔ7 SMA mice using the clasping response which is characterized by clasping of either the forepaw or hindpaw following gentle stimulation with a probe. Clasping responses were elicited in neonates at PND02 until PND08. At all ages tested, every mouse irrespective of genotype elicited a clasping reaction in response to tactile stimulation of either the forelimb or the hindlimb (data not shown).
The visual placing response was used to assess visual sensory function in SMNΔ7 SMA mice. For visual placing, a mouse is suspended by its tail and observed for the grasping of an object placed within their field of vision. In order for this response to occur, the eyelids must first be able to open. All of the SMNΔ7 carrier and normal mice were able to open their eyelids. Of those SMNΔ7 SMA pups that lived to PND13, 52.6% (10/19) were able to open their eyelids (Mann-Whitney U = 160, p < 0.001 when comparing SMA to carrier mice). Eyelid opening occurs on average at PND14 for carrier and normal mice (14.0 ± 0.4 days for either genotype) whereas the average onset of eyelid opening occurred later in SMNΔ7 SMA mice (15.2 ± 0.4 days for SMA mice; p < 0.001). Visual placing response was measured on the day following eyelid opening (i.e. PND13-15). All of the SMNΔ7 carrier and normal mice elicited a response to visual stimulation while only 10% (1/10; U = 16.0, p < 0.001 when comparing SMA to carrier mice) of those SMNΔ7 SMA who were able to open their eyelids elicited a visual placing response.
Onset of Motor Responses
Vectorial movement is defined as locomotion in one direction at a distance greater than its body length. Vectorial movement includes both crawling—movement using both limbs but being supported only with the forelimbs—and walking—movement and support with both limbs. Crawling occurs at PND04 and PND07 while walking is observed at PND11 and PND14. Nearly all of the SMNΔ7 SMA mice were not able to demonstrate vectorial movement at PND04-PND11 while one-third of the SMNΔ7 SMA mice at PND14 were able to walk (). The vectorial movement latency (; testing day F3,114 = 14.94, p < 0.001; testing day × genotype F3,114 = 4.75, p = 0.004) is significantly longer for SMNΔ7 SMA mice than for non-SMA mice at all ages tested. For SMNΔ7 SMA, the duration of movement is significantly shorter than it is for non-SMA mice (; testing day F3,114 = 69.3, p < 0.001; testing day × genotype F3,114 = 49.6, p < 0.001). Amongst non-SMA mice, movement latency decreases (F3,87 = 29.6, p < 0.001) and the movement duration increases (F3,87 = 198.4, p < 0.001) with time.
Development of motor responses in SMNΔ7 SMA mice
Spontaneous motor activity, as monitored by counting the number of grids crossed within one minute, was significantly lower in SMNΔ7 SMA mice than their non-SMA littermates at all ages tested (; testing day F3,114 = 35.0, p < 0.001; testing day × genotype F3,114 = 24.0, p < 0.001). The number of grids crossed for non-SMA mice was greater at PND11 and PND14 than at PND07 (F3,87 = 92.7, p < 0.001). SMNΔ7 SMA mice pivoted fewer times than their non-SMA littermates at all ages tested (; testing day F3,114 = 9.52, p < 0.001; testing day × genotype F3,114 = 5.47, p = 0.002). The number of pivots observed increases with age in non-SMA mice (F3,87 = 25.1, p < 0.001) but does not change with age in SMNΔ7 SMA mice.
Most SMNΔ7 SMA mice were unable to successfully complete the homing test (success rate at PND11 = 5.6% and at PND14 = 16.7%; ) within the allotted time (3 min) whereas most of the aged-matched non-SMA littermates were able to complete the homing test. The time required to complete the homing test was significantly longer for SMNΔ7 SMA mice at both ages tested (). For non-SMA mice, the homing test latency is shorter at PND14 than at PND11 (F1,48 = 41.4, p < 0.001). The homing test latencies for non-SMA mice were shorter at PND14 than at PND11. In agreement with the spontaneous motor activity results, SMNΔ7 SMA mice crossed fewer grids during the homing test than their non-SMA littermates ().
Very few (9.1%) SMNΔ7 SMA mice were able to splay their hindlimbs () at PND11 (Mann-Whitney U < 0.001, p < 0.001 when comparing SMA to carrier mice) and PND14 (U = 5.5, p < 0.001 when comparing SMA to carrier mice) whereas almost all of the non-SMA mice tested demonstrated hindlimb splay. The proportion of SMNΔ7 SMA mice that were able to grasp a wire mesh () was significantly lower than that for non-SMA mice at PND11 (9.1%; U = 41.0, p = 0.006 when comparing SMA to carrier mice) and PND14 (9.1%; U = 8.0, p < 0.001). The amount of time the mouse was able to grasp the wire mesh when suspended upside down (hanging time) is used as a measure of grip strength. As expected, the hanging time is significantly shorter for SMNΔ7 SMA mice () than for non-SMA littermates at PND11 (p = 0.011) and at PND14 (p = 0.002). The hanging time is not different between carrier and normal mice (p = 0.117 at PND14).
Use of Behavioral Characteristics to Identify SMNΔ7 SMA Mice
To determine if SMNΔ7 SMA mice could be accurately identified by phenotype, two motor behaviors were selected—surface righting reflex and negative geotaxis—based on their rapid assay times. There is a significant association between the absence of a righting reflex and the SMA genotype at PND02 (; χ2
= 36.425; p < 0.001
= 0.666, p < 0.001) as well as at PND04 (χ2
= 63.622; p < 0.001
= 0.881, p < 0.001). The accuracy of identifying SMA mice by righting reflex improves at PND04 (odds ratio of SMA mouse not being able to exhibit righting reflex = 399) from PND02 (odds ratio = 32.8). The number of SMA mice that are incorrectly identified as non-SMA based on righting reflex reduces with age (frequency of false negatives—that is, the number of SMA mice that are able to right themselves—at PND02 is 0.292 and at PND04 is 0.125). As with righting reflex, the absence of a negative geotaxis response is strongly associated with the SMA genotype () at both PND04 (χ2
= 16.98; p < 0.001
= 0.464, p < 0.001). and PND08 (χ2
= 48.84; p < 0.001
= 0.786, p < 0.001). Using negative geotaxis response as a means of identifying SMA mice is more accurate at PND08 (odd ratio = 100.7) than at PND04 (odds ratio = 9.3); however, identification of SMA mice by the absence of a righting reflex response is more accurate and can be tested earlier than that by the absence of a negative geotaxis response.
Use of righting reflex as a means to identify SMA mice from non-SMA littermates
Use of negative geotaxis as a means to identify SMA mice from non-SMA littermates
Phenotype characterization of SMNΔ7 SMA mice could potentially be used as a way to screen the effectiveness of a therapeutic agent. We used power analysis to determine the minimum number of SMNΔ7 SMA mice that would be required to observe a significant rescue of various phenotypes. The sample sizes were calculated based on α = 0.05 and a power of 80% (). For example, only one mouse would be required to observe a doubling (100% increase) in survival while 16 mice would be need to see a 20% increase in lifespan. In order to see a 100% increase in righting reflex latency at PND02, 11 mice would be required. Those phenotypes which have a greater degree of variability (such as walking latency) tend to require more mice.
Power analysis of various phenotypes in SMNΔ7 SMA mice