This study was conducted on 15 kittens (10 females and 5 males) from 6 consecutive litters that were born and raised in a closed breeding colony at Dalhousie University over a 2-year period that began in September 2022. All rearing and experimental procedures complied with ARRIVE standards. Experiments were conducted in accordance with protocols approved by the University Committee on Laboratory Animals at Dalhousie University that conformed to guidelines from the Canadian Council on Animal Care. Experiential rearing and visual testing occurred during the period from birth to 4 months of age during which time animals were housed in enriched colony rooms that were illuminated under a 12:12 h light/dark cycle. Prior to being weaned at around 2 months of age, kittens were housed together with the mother in a large space with open enclosures. Animals were fed dry cat chow (Loblaws, Toronto, Canada) ad lib and received 80 g of wet commercial cat food each day (Loblaws, Toronto, Canada; Nestle Purina, Mississauga, Canada). Behavioural tests that we employed did not require any reduction in the amount or nature of their daily food intake. Following this study, two animals remained in the colony to expand the breeding colony while all others were adopted as house pets by members of the university community after being neutered or spayed by the university veterinarian.

Each kitten was assigned to one of three groups at around 2 weeks of age in a quasi-random order made necessary by the marked differences in litter size (from 1 to 5 kittens). The rearing history of each kitten from the six litters is listed in Tables 1, 2 together with the final acuities attained by their two eyes (Table 1). Figure 1 provides a visual aid to the rearing history of the three groups that consisted of a control group (MD only, N = 2 kittens) and two experimental groups that received MD followed by either 1 (1 x RO; N = 7) or 2 (2 x RO; N = 6) periods of reverse occlusion. Because all but two of the litter sizes were small it was decided to concentrate upon population of the two experimental groups, a decision made comfortable by the fact that data was available from past studies (Duffy and Mitchell, 2013; Mitchell et al., 2016) conducted in this laboratory with animals reared similarly to those in the control group. All animals received an initial 7-day period of MD by eyelid suture of the left eye from postnatal day 21 to day 28 (P21-28). With the exception of the two control animals that received no further experiential manipulation, the remaining animals received either a single (1 x RO group; N = 7) or else two (2 x RO group; N = 6) periods of reverse occlusion. One of the periods of reverse occlusion occurred at either 5 or 6 weeks of age for all animals and lasted for 2 (N = 2) or 3 weeks (N = 11). For the 7 animals assigned to the (1 x RO) group, their single period of RO occurred at this age. For the 6 kittens assigned to the other (2 x RO) group, a short 3-day period of reverse occlusion was imposed either immediately (animals PT1-2, PT4-7, PT4-8, PT6-13, and PT6-14) or 3 days after (kitten PT5-10) the initial period of MD that was terminated at about 4 weeks of age.

All surgical manipulations necessary for the initial monocular deprivation and for the subsequent periods of reverse occlusion were conducted by KRD or DEM. As with our customary practice (Duffy et al., 2014), animals were monocularly deprived under general gaseous anesthesia (3–4% isoflurane in oxygen) in a two-stage procedure. Preceding the surgical procedures, local anaesthesia was ensured by application of Alcaine sterile ophthalmic solution (1% proparacaine hydrochloride; CDMV Canada) to the palpebral conjunctivae. Closure of the upper with the lower palpebral conjunctivae with sterile 5–0 vicryl suture thread was followed by closure of the eyelids with 5–0 silk sutures. Upon completion of surgery, animals were administered oral Metacam (meloxicam 0.05 mg/kg) for post-procedure analgesia and they also received a topical broad-spectrum ophthalmic antibiotic (1% chloromycetin; CDMV Canada) to mitigate against post-surgical infection. The quality of eyelid closure was monitored closely on a daily basis to ensure that the eyelids remained fully closed. Termination of eyelid closure was achieved under gaseous anesthesia by removal of the conjunctival and eyelid sutures and followed the same post-surgical procedures as described for the initial eyelid closure. With the exception of 4 of 5 kittens in one litter (Litter 6) the periods of eyelid closure were observed as complete throughout their intended durations. As described in detail in the Results section, the eyelids of 4 kittens in litter 6 were observed to be open partially for a time during the intended period of occlusion.

Training and behavioural testing of grating acuity employed a jumping stand and followed procedures outlined in earlier papers (Mitchell et al., 1977; Murphy and Mitchell, 1987, Mitchell, 2013 and upgraded recently Mitchell, 2025). Visual acuity was measured longitudinally with one experimenter seated on either side of the jumping platform to minimize the emergence of a side preference and to ensure consistent behaviour across all animals over the 2 years necessary to collect the data. The method capitalizes upon a natural behaviour of kittens to descend by scrambling or jumping for a food or petting reward to one of two large adjacent square-wave grating patterns having the same period but at right-angles to each other beneath them. The positive (rewarded) stimulus was a vertical grating while the unrewarded grating was horizontal. Jumps to the latter were not rewarded and the animals were required immediately to repeat the trial. The jumping platform from which the kitten jumped was supported by two yoked laboratory jacks to enable small and continuous changes in its distance to the grating stimuli in a manner commensurate with the kitten’s ability to jump at any age. Training began on the jumping stand at 4–5 weeks of age and was usually completed in a few days.

Visual acuity for square-wave gratings were measured by a strictly descending method of limits with small decrements in stimulus magnitudes (in this case, spatial frequency) that were equated on a logarithmic scale with as many as 12 steps to an octave. In order to constrain the total number of trials the spatial frequency was increased after only one or two successful trials but, closer to threshold, the minimum number of trials was increased to five. After an error, the animal was denied the food reward and was required to repeat the trial. Once an error had been corrected, the animal was required to make five consecutively correct responses or else make seven correct responses out of a maximum of 10 trials at any spatial frequency before proceeding to the next higher spatial frequency. The threshold was defined as the highest spatial frequency for which this criterion performance level was achieved. It was typical for animals to perform flawlessly until near threshold where, in addition to errors, they would exhibit general signs of difficulty with its choice that included marked increased latency, vocalizations and variation of its patterns of gaze from the stimuli themselves to other objects in the room. The nervousness and reluctance exhibited by most kittens at the highest grating spatial frequencies prevented the use of a strict staircase procedure that incorporates trials with stimuli of progressively lower spatial frequency in the immediate aftermath of this failure. When possible, additional estimates of acuity in a single session were obtained by repetitions of a descending method of limits but starting with gratings of a different spatial frequency (usually higher) than that employed for the immediately prior assessments.

Measurement of the grating acuities of the two eyes were conducted on all kittens until they were between 3 and 4 months old at which age grating acuity as measured on the jumping stand has been shown to achieve asymptotic values (Mitchell et al., 1976). Using procedures previously described (Hogan et al., 2023), assessment of the refractive status of each eye of all 11 kittens of 3 litters (PT4, PT5, PT6) by retinoscopy at the conclusion of testing revealed that they were either emmetropic or within 0.25D of this status in each eye.

Although the mean visual acuity recovered by the deprived eye of animals that received two temporally separated periods of reverse occlusion was significantly lower than that for animals that received only a single period of such treatment, the size of the improvement in both groups was low. In retrospect, the outcome of this study may have been more substantial if the late (i.e., second) period of reverse occlusion had been introduced earlier in life so as to increase its potential efficacy. However, the constraints inherent with the use of cats from a laboratory breeding colony of limited size to model amblyopia, such as small and varying litter sizes and irregular pregnancies, required commitment to the original choice of experimental design until completion of the study. Nonetheless, the behavioral outcomes of this study are supported by previous electrophysiological and anatomical studies on cats and monkeys that revealed interactions between temporally adjacent experiential manipulations. In particular, these past studies reveal that an early episode of abnormal visual experience alters the effect magnitude of an interlude of similar or different anomalous visual exposure imposed at a later age (Mustari and Cynader, 1981; Faulkner et al., 2005; Smith et al., 1992; Henneberry et al., 2023).

The first of these studies was conducted on kittens in the laboratory of a past departmental colleague (Mustari and Cynader, 1981) and demonstrated that an early period of ocular misalignment (strabismus) attenuated the electrophysiological effects of an 8-day period of MD in the visual cortex imposed at P45 days as compared to that observed in response to the same deprivation imposed on normal kittens at the same age. A similar conclusion was reached 2 decades later by use of optical imaging of intrinsic signals to compare the extent of cortical territory dominated by each eye following a 10-day period of MD imposed on two groups of cats (Faulkner et al., 2005). Whereas control animals received a single 10-day period of MD at P35, animals in the experimental group had been made strabismic by myotomy of one of the extraocular muscles 3 weeks prior to imposition of the same 10-day period of MD at 35 days of age. Longitudinal measurement of the rate of ocular dominance recovery after the period of MD was found to be both slower and less complete in the animals that had been made strabismic before the period of MD. A similar finding was observed in monkeys where prior binocular dissociation via strabismic rearing reduced the efficacy of MD to produce form deprivation amblyopia (Smith et al., 1992).

That an early transitory period of MD might also produce changes in the visual cortex that influence the response to such deprivation induced much later was demonstrated in mice by the discovery that such early MD promoted an augmented response to imposition of a second period of MD imposed on the same eye (but not the fellow eye) in adulthood (Hofer et al., 2006; Hofer et al., 2009). The broader possibility that a brief early period of monocular deprivation might alter the response to a second interval of such exposure imposed shortly afterward was explored in a recent extensive anatomical and electrophysiological study conducted on kittens (Henneberry et al., 2023). Notably, it was demonstrated that an early short period of MD reduced substantially the anatomical and electrophysiological responses to a subsequent period of occlusion or retinal inactivation of the fellow eye in comparison to that observed in control animals that received only the latter deprivation.

Further insight into the consequences of temporally spaced early experiential interventions are available from studies conducted on kittens and infant monkeys that were reared with repeated daily episodes of typical vision followed or preceded by intervals of grossly abnormal visual exposure (Mitchell et al., 2003; Wensveen et al., 2006). These studies also provided information on the closely related issue of the extent to which the impact of separate daily episodes of visual exposure were additive. The monkeys for these studies were reared with helmets that were worn continuously from 3 to 18 weeks of age and enabled either binocular clear viewing through plano lenses in front of both eyes for part of each day followed or preceded by monocular viewing with a diffuser lens in front of one eye and a plano lens in front of the other eye. For kittens, opaque neoprene masks were used to enable the daily period of monocular visual exposure and were removed for the episode of binocular exposure. The masks required close supervision by a technician to ensure they were worn and consequently the total daily visual exposure was restricted to 7 h each day. For the remaining 17 h each day the kittens were housed in complete darkness as described in detail elsewhere (Mitchell, 2013). An additional difference between the monkey and kitten studies was in regard to the methods of behavioral testing which for the kittens permitted immediate evaluation of the visual acuity of each eye at the conclusion of the period in which they received mixed daily visual exposure. By contrast, the long behavioral training necessary for the monkeys meant that this information was obtained a year following the period of restricted daily visual exposure. Despite the substantial differences in the rearing and testing procedures for the two species, results were similar in that 2 h of daily normal visual exposure was sufficient to offset much longer periods of daily monocular exposure to allow development of normal acuity in both eyes (Mitchell et al., 2003; Wensveen et al., 2006). Importantly it was shown in kittens that two separated periods of binocular exposure were not necessarily additive and in certain circumstances one of the two binocular instalments appeared to be completely ineffective (Mitchell et al., 2006, see Fig. 5). The latter result is also consistent with the current data indicating that two separated episodes of occlusion treatment produced significantly less recovery in comparison to just one.

As summarized above, the non-additive consequences of the two temporally separated occlusion treatments administered to the amblyopic animals of our study add to a body of pre-existing data derived from animals concerning the consequences of separated episodes of selected early visual exposure. Our findings, when combined with the results of these prior studies hold important implications for clinical treatment of amblyopia in humans.

Notwithstanding the reservations expressed earlier concerning the duration and spacing of the particular visual exposures employed in the present study, the results reached statistical significance. In confirmation of earlier results from research on animal models of amblyopia, it is apparent that an early or brief period of occlusion of the non-amblyopic eye may subsequently have unexpected visual consequences that may even include the emergence of bilateral amblyopia (Mitchell et al., 1984; Murphy and Mitchell, 1987). In combination, results from these animal studies suggest that in addition to existing advice that experiential treatment of amblyopia such as patching be initiated early, it should also follow an exact protocol in terms of adherence to the particular treatment and be applied for a specified time. The combined components of this treatment imply a “Three-E” protocol: Early, Exact and Enduring. In addition to the existing advice for early experiential intervention the specific treatment regimen should be monitored closely with respect to adherence and thus be exact and in addition, it should be enduring with respect to its duration so as to maximize the potential efficacy. Our study points to the need for perseverance with a particular protocol in order to maximize its potential effectiveness and also provides a note of caution with respect to the potential consequences of interruption of treatment.