We developed a reliable imaging and quantitative analysis method for in vivo corneal confocal microscopy in rodents and used it to determine whether models of type-1 diabetes replicate the depletion of corneal nerves reported in diabetic patients. Quantification was reproducible between observers and stable across repeated time points in two rat strains. Longitudinal studies were performed in normal and streptozotocin-diabetic rats, with innervation of plantar paw skin quantified using standard histological methods after 40 weeks of diabetes. Diabetic rats showed an initial increase, then a gradual reduction in occupancy of nerves in the sub-basal plexus so that values were significantly lower at week 40 (68±6%) than age-matched controls (80±2%). No significant loss of stromal or intra-epidermal nerves was detected. In a separate study, insulin was applied daily to the eye of control and streptozotocin-diabetic mice and this treatment prevented depletion of nerves of the sub-basal plexus. Longitudinal studies are viable in rodents using corneal confocal microscopy and depletion of distal corneal nerves precedes detectable loss of epidermal nerves in the foot, suggesting that diabetic neuropathy is not length dependent. Loss of insulin-derived neurotrophic support may contribute to the pathogenesis of corneal nerve depletion in type 1 diabetes.

Using non-invasive, near-infrared spectroscopy we have previously reported optical signals measured at or around peripheral nerves in response to their stimulation. Such optical signals featured amplitudes on the order of 0.1% and peaked about 100 ms after peripheral nerve stimulation in human subjects. Here, we report a study of the spatial and spectral dependence of the optical signals induced by stimulation of the human median and sural nerves, and observe that these optical signals are: (1) unlikely due to either dilation or constriction of blood vessels, (2) not associated with capillary bed hemoglobin, (3) likely due to blood vessel(s) displacement, and (4) unlikely due to fiber-skin optical coupling effects. We conclude that the most probable origin of the optical response to peripheral nerve stimulation is from displacement of blood vessels within the optically probed volume, as a result of muscle twitch in adjacent areas.

We have examined ten human subjects with a previously developed instrument for near-infrared diffuse spectral imaging of the female breast. The instrument is based on a tandem, planar scan of two collinear optical fibers (one for illumination and one for collection) to image a gently compressed breast in a transmission geometry. The optical data collection features a spatial sampling of 25 points/cm2 over the whole breast, and a spectral sampling of 2 points/nm in the 650–900 nm wavelength range. Of the ten human subjects examined, eight are healthy subjects and two are cancer patients with unilateral invasive ductal carcinoma and ductal carcinoma in situ, respectively. For each subject, we generate second-derivative images that identify a network of highly absorbing structures in the breast that we assign to blood vessels. A previously developed paired-wavelength spectral method assigns oxygenation values to the absorbing structures displayed in the second-derivative images. The resulting oxygenation images feature average values over the whole breast that are significantly lower in cancerous breasts (69 ± 14%, n = 2) than in healthy breasts (85 ± 7%, n = 18) (p < 0.01). Furthermore, in the two patients with breast cancer, the average oxygenation values in the cancerous regions are also significantly lower than in the remainder of the breast (invasive ductal carcinoma: 49 ± 11% vs 61 ± 16%, p < 0.01; ductal carcinoma in situ: 58 ± 8% vs 77 ± 11%, p < 0.001).

Previously we have reported a near-infrared optical response in the region occupied by a peripheral nerve that is distal to the site of electrical stimulation of that peripheral nerve. This “intermediate” signal is vascular in nature but its biological origin not been elucidated. In the present study, an animal model of the signal has been created and our human studies expanded to directly investigate the contribution of non-artifactual vascular motion induced by muscle contraction to the biological origin of this signal. Under non-invasive conditions during stimulation of the exposed sciatic nerve of the Sprague-Dawley rat, optical responses are robust. These signals can be abolished both pharmacologically and surgically using methods that eliminate muscle motion while leaving the electrophysiological health of the nerve intact. In human studies, signals that are elicited on stimulation of nerves containing motor axons, both within and outside the predicted imaging volume of the spectrometer, have similar temporal characteristics of those previously observed. Moreover, stimulation of sensory nerves alone does not elicit an optical response. These results strongly suggest that the intermediate signals are derived from stimulus-induced muscle contraction (whether via an innervating nerve or by direct stimulation) causing translational vascular motion within the optically interrogated region.

We report our studies on the optical signals measured non-invasively on electrically stimulated peripheral nerves. The stimulation consists of the delivery of 0.1 ms current pulses, below the threshold for triggering any visible motion, to a peripheral nerve in human subjects (we have studied the sural nerve and the median nerve). In response to electrical stimulation, we observe an optical signal that peaks at about 100 ms post-stimulus, on a much longer time scale than the few milliseconds duration of the electrical response, or sensory nerve action potential (SNAP). While the 100 ms optical signal we measured is not a direct optical signature of neural activation, it is nevertheless indicative of a mediated response to neural activation. We argue that this may provide information useful for understanding the origin of the fast optical signal (also on a 100 ms time scale) that has been measured non-invasively in the brain in response to cerebral activation. Furthermore, the optical response to peripheral nerve activation may be developed into a diagnostic tool for peripheral neuropathies, as suggested by the delayed optical signals (average peak time: 230 ms) measured in patients with diabetic neuropathy with respect to normal subjects (average peak time: 160 ms).