Restoration of Contralateral Representation in the Mouse Somatosensory Cortex after Crossing Nerve Transfer

Avulsion of spinal nerve roots in the brachial plexus (BP) can be repaired by crossing nerve transfer via a nerve graft to connect injured nerve ends to the BP contralateral to the lesioned side. Sensory recovery in these patients suggests that the contralateral primary somatosensory cortex (S1) is activated by afferent inputs that bypassed to the contralateral BP. To confirm this hypothesis, the present study visualized cortical activity after crossing nerve transfer in mice through the use of transcranial flavoprotein fluorescence imaging. In naïve mice, vibratory stimuli applied to the forepaw elicited localized fluorescence responses in the S1 contralateral to the stimulated side, with almost no activity in the ipsilateral S1. Four weeks after crossing nerve transfer, forepaw stimulation in the injured and repaired side resulted in cortical responses only in the S1 ipsilateral to the stimulated side. At eight weeks after crossing nerve transfer, forepaw stimulation resulted in S1 cortical responses of both hemispheres. These cortical responses were abolished by cutting the nerve graft used for repair. Exposure of the ipsilateral S1 to blue laser light suppressed cortical responses in the ipsilateral S1, as well as in the contralateral S1, suggesting that ipsilateral responses propagated to the contralateral S1 via cortico-cortical pathways. Direct high-frequency stimulation of the ipsilateral S1 in combination with forepaw stimulation acutely induced S1 bilateral cortical representation of the forepaw area in naïve mice. Cortical responses in the contralateral S1 after crossing nerve transfer were reduced in cortex-restricted heterotypic GluN1 (NMDAR1) knockout mice. Functional bilateral cortical representation was not clearly observed in genetically manipulated mice with impaired cortico-cortical pathways between S1 of both hemispheres. Taken together, these findings strongly suggest that activity-dependent potentiation of cortico-cortical pathways has a critical role for sensory recovery in patients after crossing nerve transfer

Genetic Organization and Mode of Action of a Novel Bacteriocin, Bacteriocin 51: Determinant of VanA-Type Vancomycin-Resistant Enterococcus faecium▿†

Bacteriocin 51 (Bac 51) is encoded on the mobile plasmid pHY (6,037 bp), which was isolated from vancomycin-resistant Enterococcus faecium VRE38. Bacteriocin 51 is active against E. faecium, E. hirae, and E. durans. Sequence analysis of pHY showed that it encodes nine open reading frames (ORFs) from ORF1 to ORF9 (in that order). Genetic analysis suggested that ORF1 and ORF2, which were designated bacA and bacB, respectively, are the bacteriocin and immunity genes. bacA encodes a 144-amino-acid protein. The deduced BacA protein has a typical signal sequence at its amino terminus, and a potential signal peptidase-processing site corresponding to the V-E-A sequence is located between the 37th and 39th amino acids. The predicted mature BacA protein consists of 105 amino acids. A potential promoter sequence was identified upstream of the start codon. bacB encodes a 55-amino-acid protein. No obvious promoter or terminator sequence was identified between bacA and bacB. Northern blot analysis of bacA and bacB with a bacA RNA probe produced a transcript of approximately 700 nucleotides, which corresponded to the combined nucleotide sizes of bacA and bacB, indicating that transcription was initiated from the promoter upstream of bacA, continued through bacB, and was terminated at the terminator downstream of bacB. The transcription start site was determined to be the T nucleotide located 6 nucleotides downstream from the −10 promoter sequence. These results indicate that bacA and bacB constitute an operon and that bacA is the bacteriocin structural gene while bacB is the immunity gene. The purified C-terminally His tagged BacA protein of Bac 51 showed bacteriostatic activity against the indicator strain. The purified C-terminally His tagged BacA protein of Bac 32 (whose mature BacA protein has 54 amino acids) and the culture filtrates of the Bac 31- and Bac 43-producing E. faecalis strain FA2-2 showed bactericidal activity. Bac 31 and Bac 43 are pore-forming bacteriocins, unlike the newly characterized bacteriocin Bac 51

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

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

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 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