Tomographic optical imaging of cortical responses after crossing nerve transfer in mice

<div><p>To understand the neural mechanisms underlying the therapeutic effects of crossing nerve transfer for brachial plexus injuries in human patients, we investigated the cortical responses after crossing nerve transfer in mice using conventional and tomographic optical imaging. The distal cut ends of the left median and ulnar nerves were connected to the central cut ends of the right median and ulnar nerves with a sciatic nerve graft at 8 weeks of age. Eight weeks after the operation, the responses in the primary somatosensory cortex (S1) elicited by vibratory stimulation applied to the left forepaw were visualized based on activity-dependent flavoprotein fluorescence changes. In untreated mice, the cortical responses to left forepaw stimulation were mainly observed in the right S1. In mice with nerve crossing transfer, cortical responses to left forepaw stimulation were observed in the left S1 together with clear cortical responses in the right S1. We expected that the right S1 responses in the untreated mice were produced by thalamic inputs to layer IV, whereas those in the operated mice were mediated by callosal inputs from the left S1 to layer II/III of the right S1. To confirm this hypothesis, we performed tomographic imaging of flavoprotein fluorescence responses by macroconfocal microscopy. Flavoprotein fluorescence responses in layer IV were dominant compared to those in layer II/III in untreated mice. In contrast, responses in layer II/III were dominant compared to those in layer IV in operated mice. The peak latency of the cortical responses in the operated mice was longer than that in the untreated mice. These results confirmed our expectation that drastic reorganization in the cortical circuits was induced after crossing nerve transfer in mice.</p></div

Conventional imaging of cortical responses after crossing nerve transfer.

<p>(A) Schematic drawing of crossing nerve transfer. IM represents imaged area. (B) Original fluorescence image. (C) Pseudo-color image of fluorescence responses elicited by vibratory stimulation at 50 Hz for 0.5 s applied to the left forepaw. The image in (B) and the responses in (C) were recorded in the same mouse with crossing nerve transfer. (D) Fluorescence responses elicited by stimulation of the left forepaw in an untreated mouse. The contralateral right S1 shows clear responses, while the ipsilateral left S1 is only weakly activated. (E) Time course of ΔF/F₀ changes in the square windows (1–4) shown in (C) and (D). (F) Amplitudes of the cortical responses. Mean and S.E.M. are shown. (G) Bilaterality index (ratio of the inferior response amplitudes normalized by the superior response amplitudes) in the untreated and operated mice.</p

Tomographic imaging of cortical responses in untreated mice.

<p>(A) Original tomographic images in the right S1 (upper panels) and pseudo-color images of fluorescence responses elicited by vibratory stimulation at 50 Hz for 0.5 s applied to the left forepaw (lower panels). The numbers in the upper panels represent the depth (μm) from the cortical surface. The two arrows in the upper panels show the position of an artery that is visible in the leftmost panel but not in the rightmost panel. The circle in the leftmost lower panel shows the circular window in which response amplitudes were measured in ΔF/F₀. (B) Time courses of the fluorescence responses measured at 50, 200, 400 and 800 μm deep from the cortical surface using a macroconfocal microscope. Data in (A) and (B) were obtained from the same mouse. (C) Amplitudes of fluorescence responses measured at each depth.</p

Tomographic imaging of cortical responses in operated mice.

<p>(A) Original tomographic images in the right S1 (upper panels) and pseudocolor images of fluorescence responses elicited by vibratory stimulation at 50 Hz for 0.5 s applied to the left forepaw (lower panels). (B) Time courses of the fluorescence responses measured at 50, 200, 400 and 800 μm deep from the cortical surface using a macroconfocal microscope. Data in (A) and (B) were obtained from the same mouse. (C) Amplitudes of fluorescence responses measured at each depth.</p

Comparison of cortical responses between untreated and operated mice.

<p>(A) Time courses of the fluorescence responses measured at 50, 200, 400 and 800 μm deep from the cortical surface using a macroconfocal microscope. Statistical differences were evaluated regarding the peak amplitude and peak latency between the untreated and operated mice. (B) Relative response amplitudes at 200 μm normalized to those at 400 μm. This ratio is smaller than 1.0 in the untreated mice, while it was larger than 1.0 in the operated mice. (C) Schematic drawing of the neural circuits in the operated mice.</p

Crossing nerve transfer in cPcdhα knockout mice.

<p>(A) Cortical responses elicited by transcranial stimulation of the left S1 in control cPcdhα +/+ (a) and cPcdhα −/− mice (b). Responses in a cortex-restricted GluN1 +/− mouse are also shown for comparison (c). (B) Bilaterality index of cortical responses elicited by transcranial stimulation of the left S1 in cPcdhα +/+, cPcdhα −/−, and cortex-restricted GluN1 +/− mice. (C) Cortical responses elicited by left forepaw stimulation at 8 weeks after crossing nerve transfer in cPcdhα +/+ (a) and cPcdhα −/− mice (b). (D) Bilaterality index after crossing nerve transfer in cPcdhα +/+ and cPcdhα −/− mice.</p

Cortical responses before and after crossing nerve transfer.

<p>(A) Cortical responses elicited by vibratory stimulation applied to the left forepaw in a naïve mouse. In the left diagram (a), peripheral nerves are shown on the back of a mouse to avoid left-right confusion. The middle panel (b) shows the original fluorescence image. In the right panel (c), neural activity is apparent in the contralateral right S1, while the ipsilateral left S1 is only weakly activated. (B) Cortical responses after crossing nerve transfer. The diagram (a) and cortical responses elicited by vibratory stimulation applied to the left forepaw at 4 weeks (b), 8 weeks (c and d), 8 months (e) and 12 months (f) after crossing nerve transfer. The cortical responses shown in (c) were almost completely lost after the nerve graft was cut in the same mouse (d).</p

Bilaterality index in mice with or without crossing nerve transfer.

<p>The bilaterality index in mice with crossing nerve transfer surgery using three different methods is significantly greater than that in naïve mice (P<0.0001, respectively). The bilaterality index is not significantly different between mice operated in an end-to-end fashion, with or without sequential nerve cut. However, the index in mice operated in an end-to-side fashion was significantly larger than that in mice operated in an end-to-end fashion with (P<0.002) or without sequential nerve cut (P<0.007). Numbers in the parentheses show the numbers of mice.</p

Bilateral cortical representation induced by direct cortical stimulation.

<p>(A) Cortical responses elicited by left forepaw stimulation before (a), within 20 min (b) and more than 40 min (c) after cessation of direct stimulation applied to the ipsilateral left S1 paired with left forepaw stimulation in the same mouse. These experiments were performed in naïve mice. The black dot in (a–c) shows the direct cortical stimulation site. (B) Amplitudes of cortical responses elicited by left forepaw stimulation in the ipsilateral left S1 (red) and contralateral right S1 (blue).</p

Crossing nerve transfer surgery.

<p>The cut ends of the left median (MN) and ulnar nerves (UN) were connected to the right brachial plexus (BP) via a sciatic nerve graft at 8 weeks of age. The left radial (RN) and musculocutaneous nerves (McN) were cut at the same time, or at 15 weeks of age (sequential nerve cut). Imaging was performed at 16 weeks of age.</p