Intrathecal Delivery of BDNF Into the Lumbar Cistern Re-Engages Locomotor Stepping After Spinal Cord Injury

Abstract

Delivery of neurotrophins to the spinal injury site via cellular transplants or viral vectors administration has been shown to promote recovery of locomotion in the absence of locomotor training in adult spinalized animals. These delivery methods involved risks of secondary injury to the cord and do not allow for precise and controlled dosing making them unsuitable for clinical applications. The present study was aimed at evaluating the locomotor recovery efficacy and safety of the neurotrophin BDNF delivered intrathecally to the lumbar locomotor centers using an implantable and programmable infusion mini-pump. Results showed that BDNF treated spinal cats recovered weight-bearing plantar stepping at all velocities tested (0.3-0.8 m/s). Spinal cats treated with saline did not recover stepping ability, especially at higher velocities, and dragged their hind paws on the treadmill. Histological evaluation showed minimal catheter associated trauma and tissue inflammation, underlining that intrathecal delivery by an implantable/programmable pump is a safe and effective method for delivery of a controlled BDNF dosage; it poses minimal risks to the cord and is clinically translational.

Fig. 1.

Experimental setup: Location of the reflective markers and diagram of the kinematic parameters evaluated. Markers were placed on the cat over the humeral head (A), the elbow joint lateral epicondyle (B), the lateral carpal joint (C), the metacarpophalangeal joint (D) and the tip of the digits (E) on the right forelimb, as well as on the level of the ischium (F), femoral head (G), knee joint (H), lateral malleolus (I), metatarsophalangeal joint (J) and the tip of the digits (K) on the right hindlimb. Some kinematic parameters are also represented: stance length, swing height, hip height, Da, Dp and hip, knee and ankle angles.

Fig. 2.

Kinematics of the right hindlimb: Stick figures represent the stance and swing phase of one step for a representative cat from each group pre-transection and 5 weeks post-injury at 0.4 m/s. The arrow indicates the direction of forward progress. Control cats showed dorsal foot placement during stance, while most BDNF treated cats were able to execute plantar weight bearing stepping at all the velocities tested (0.3-0.8 m/s).

Fig. 3.

A) and B) show the EMMs±95% CI of the stance length and swing height indices 3- and 5-weeks post-Tx at medium (0.4 m/s, black bars) and maximum (0.8 m/s, grey bars) walking velocity for the BDNF group (N = 6 animals for 0.4 m/s walking speed at 5 weeks; N = 5, 0.4 m/s at 3 weeks & 0.8 m/s at 5 weeks; N = 3, 0.8 m/s at 3 weeks). The dashed black and grey lines represent values obtained with treadmill training alone (BWST) [1], [2] and with neurotrophins cellular delivery (NTF) [3] respectively. C) and D) show the EMMs±95% CI for the Da and Dp indices for the BDNF group at the same time points and walking speeds. The dotted black line indicates pre-Tx performance (100%) in all panels. For most parameters (except Dp) the EMM 95% CIs post-Tx did not overlap with the pre-Tx levels, indicative of a remaining deficit, but were similar or above the levels obtained with BWST or NTF.

Fig. 4.

EMMs of the hip, knee and ankle joints angles for BDNF (N = 6 for Pre-Tx and 5 wks, N = 5 for 3 wks) cats during walking at 0.4 m/s for all time points: pre-transection (Pre-Tx), 3, and 5 weeks after injury. The black bars represent the hip, knee and ankle angles range, the light grey bars represent the joints maximum angle and the dark grey bars show the values of the minimum joints angle. All values are reported as EMM±95%CI. Linear mixed models with the angular parameter as dependent variable and time as a repeated measure factor showed a significant effect of time for all the parameters except the ankle joint minimum angle. Post-hoc EMM pairwise comparisons (all time points) with Bonferroni correction showed a number of significant differences with pre-Tx values for the post-Tx joint angle parameters (indicated by * along with the p-value) but no significant differences between angular parameters at 3 and 5 weeks post-Tx.

Fig. 5.

Profiles of the normalized vertical GRFs of the right hindlimb over multiple steps in an exemplar 10 seconds period for one representative cat of each group, Control A) and BDNF B). Although the average peak vertical forces during a trial were lower than 4 N/kg (dotted horizontal line), BDNF treated cats were capable of full weight bearing stepping over some of the stepping period. On the other hand, Control cats did not display any ability to full weight support during the trial, with peaks of the vertical forces lower than 2-2.5 N/kg. C) shows the EMMs ± 95%CI of the peak vertical forces normalized for the animals’ weight at the moment of the recordings under the right and left paws. The black bars refer to BDNF treated cats (N = 4) while the grey bars to the Control group (N = 2 that could execute some plantar steps). The dotted black line represents the minimum required vertical force needed for full hindlimb weight support (about 4 N/kg). BDNF treated cats showed significantly higher weight support when compared to Control cats (see text for details) although the 95% CI EMMs of the peak vertical forces overlapped for the left limb (lack of overlap for the right limb is indicated by joining bars).

Fig. 6.

GFAP expression at the lesion site A) and at the catheter insertion area B): Staining was performed on eight animals, 5 BDNF (2 with L3 delivery and 3 with L7 delivery) and 3 Controls (two with L3 delivery and one with L7 delivery) on horizontal sections taken from the catheter entry zone’s segment (L7/S1) to the segment where the tip of the catheter was found post-mortem. We also stained the transection site of four cats (3 BDNF and 1 Control) to serve as control/reference. A) The staining at the lesion site of BDNF-treated cat, going from rostral (R) to caudal (C) of the lesion, indicates glial reactivity of the tissue surrounding the injury. Image at higher magnification show enlarged GFAP positive cells. B) Catheter insertion area of a BDNF cat. The catheter was found on the ventrolateral aspect of the cord in the L7 segment (yellow arrow). We did not find any evidence (indentation or staining) on the cord of the exact location of the catheter tip, indicating that no damage to the cord was caused by catheter insertion. GFAP staining confirmed this visual observation as minimal to no evidence of over background GFAP levels occurs at the catheter insertion area. In (B) the higher red expression observed at the spinal cord border (white arrows) are astrocyte along the spinal cord pia, which often show higher levels of GFAP staining. This edge staining pattern was observed along the entire length of the spinal cord. Higher magnification image on the left shows stronger GFAP staining within the astrocytes along the pial margin.