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Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury. However, its effect on spinal cord injury in aged mice remains unclear. Considering the essential role of angiogenesis during the regeneration process, we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells, thereby promoting microvascular regeneration in aged mice after spinal cord injury. In this study, we established young and aged mouse models of contusive spinal cord injury using a modified Allen method. We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord. Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro. Furthermore, intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord, thereby improving neurological function. The role of metformin was reversed by compound C, an adenosine monophosphate-activated protein kinase inhibitor, both in vivo and in vitro, suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury. These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway, thereby improving the neurological function of aged mice after spinal cord injury.

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O'Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O'Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 - 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 - 13.6 × 2.2 - 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.

Sea buckthorn (Hippophae rhamnoides) is an important deciduous shrub for fruit and ecological restoration in arid and semi-arid regions of China. Twelve Chinese and Russian cultivars (cv. Shenqiuhong, eshi01, ... eshi11) were planted about 1.6 acre area in a seedling nursery, located in Qingyang City of Gansu province in northwest China, where high mortality (more than 70%) of sea buckthorn was observed in late July 2019. Symptoms consisted of massive chlorosis, drooping leaves and dried-up stems on 5-year-old trees. Pieces of tree roots and stems with irregular light-brown discoloration in the xylem vessels were selected. Small pieces of discolored tissue were surface disinfested (1 min in 1% sodium hypochlorite, followed by three rinses with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues. Colonies on PDA grew rapidly. Dense mycelia were pinky-white initially, and became carmine red color with age on the undersurface of the plate. Macroconidia were moderately curved, 3 to 5 marked septa, hyaline, thick walled, and measuring 27.8± 3.6 µm × 4.8 ± 0.5 µm (n = 30). Microconidia were abundant, pear-shaped, ellipsoid to fusoid, often with a papilla at the base, and 8.4 ± 2.2 µm ×3.1 ± 0.3 µm (n = 30). Genomic DNA was extracted for amplification and sequencing of the internal transcribed spacer region (ITS1 and ITS4 primers) (White et al. 1990) of the ribosomal DNA (Accession Nos. MN160235 to MN160238) and translation elongation factor-1 alpha (EF1 and EF2 primers, accession Nos. MN429075 to MN429078) (O'Donnell et al. 1998). The sequences revealed 99% similarity to the sequences of the ITS (AY188917), and 100% identity with EF1-α (JF740808) regions of Fusarium sporotrichioides. Based on morphological and molecular characteristics, the fungus was identified as F. sporotrichioides (Leslie and Summerell 2006). Koch's postulates were fulfilled on healthy, potted 1-year-old sea buckthorn seedings using two isolates in a greenhouse at 25 °C, 90% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedings were inoculated with PDA plugs as controls. Seven to ten days after inoculation, typical symptoms of dark-brown necrotic lesions on chlorotic leaf margins were observed. About 2 weeks after inoculation, the inoculated stems were gradually dry up, accompanied by withering and fallen leaves. Control plants remained asymptomatic. Pathogens were successfully isolated from the inoculated stems again, exhibiting morphological characteristics identical to those of F. sporotrichioides. Previous papers reported F. sporotrichioides as a common pathogen caused lavender wilt (Cosic et al. 2012), foliar spots on forage corn (Moya-Elizondo et al. 2013) and maize ear rot (Wang et al. 2019). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. sporotrichioides on several Chinese and Russian cultivars in Gansu province of China. In Heilongjiang province, the same disease was reported in 2010 (Song et al. 2010), nearly 30 longitudes away from Gansu province. Therefore, this disease appears to be a serious risk for future sea buckthorn production.