a, b, Baseline freezing levels (a) and cued fear-conditioning freezing levels (b) in young WT mice treated with anti-TIMP2 IgG or control IgG (60 μg kg−1) for 2 weeks (n = 15 per group; 2-month-old). c, d, Serum metabolite measurements (c) and weekly weights (d) to assess general health and organ function in young WT mice treated for ~4 weeks with anti-TIMP2 IgG or control IgG. e, f, Proportion of trial time spent in centre (e) and velocity in centre (f) during open-field assessment of anxiety-like behaviour in the treated mice. g–i, Velocity in zone outside the centre (g), total trial distance (h), and mean trial velocity (i; centre and outside) during open-field testing. j–m, General activity (j), rearing activity (k), distance travelled (l), and mean trial velocity (m)—all monitored by SMARTCage beam-breaks in a home cage for the treated mice. n, o, Quantification of total newborn neuron number in dentate gyrus (n) with corresponding representative dentate gyrus sections stained with doublecortin antibody (o; DCX, arrowheads; scale bar, 100 μm) in control IgG- or anti-TIMP2 IgG-treated young mice; For d–o, n = 15 mice per group; 2.5-month-old; in c, one serum sample in each group was not submitted for metabolite testing owing to a high degree of haemolysis in these two samples; Student’s t-test; *P
a, Table of significant changes (ranked by effect size) in the relative level of plasma proteins in young (3-month-old) TIMP2 KO (n = 13) versus WT (n = 9) mice measured by customized protein microarray. The first two entries represent two different antibodies against TIMP2. b, Mean TIMP2 concentrations determined by ELISA (±s.d. for technical replicates) for cord plasma pre-depletion, or cord plasma after TIMP2 or control depletion. c, Silver-stained gel loaded with elution from beads used for TIMP2 (T2) or control (ctl) depletion. ‘#1’ and ‘#2’ reflect replicate plasma aliquots from the depletion process (see Supplementary Fig. 1 for uncropped gel image). d, e, Baseline freezing levels (d) and cued fear-conditioning freezing levels (e) in aged NSG mice (13.8 ± 0.1 months old) given eight intravenous injections of vehicle (n = 10), TIMP2-depleted cord plasma (n = 8), and IgG control-depleted cord plasma (n = 9). f, g, Proportion of trial time spent in centre (f) and velocity in centre (g) during open-field assessment of anxiety-like behaviour in the treated mice. h–j, Velocity in zone outside the centre (h), total travel distance (i), and mean trial velocity (j; centre and outside) during open-field testing. k–m, General activity (k), distance travelled (l), and mean trial velocity (m) by SMARTCage beam-break monitoring for the treated mice. One-way ANOVA with Tukey’s post hoc test; Student’s t-test for WT versus TIMP2 KO comparisons with q
Figure 1. Human cord plasma enhances plasticity…
Figure 1. Human cord plasma enhances plasticity in the aged brain
a , Human plasma…
Figure 1. Human cord plasma enhances plasticity in the aged brain a, Human plasma (PLM) transfer protocol in aged NSG mice. b, Heat map of unsupervised clustering of gene changes in hippocampi following human plasma treatment of aged NSG mice (n = 7 per group; 13.8 ± 0.2 months old; colour bar represents Z-score range; one-way analysis of variance (ANOVA); P < 0.05). c, Three-dimensional plot of the first three components from PCA of whole-genome microarrays (WGM); inset graph represents eigenvalues (y axis) plotted against component number (x axis). d, Relative gene expression by qPCR for confirmatory plasticity genes after vehicle or human cord plasma treatment (n = 7 per group; 13.9 ± 0.2 months old). e, f, c-Fos-positive cell number in dentate gyrus (DG) of human-plasma-treated aged (e; n = 8, 7, 7, 7 mice per group (left to right); 13.9 ± 0.2 months old) or young (f) NSG mice (n = 8 mice per group; 3.4 ± 0.1 months old). g, Representative dentate gyrus and CA1 images and (h, i) recombined cell quantification (red; TdTomato) with counterstain (green; TO-PRO-3) from treated TRAP-FOS mice (n = 4, 3, 4 mice per group (left to right); 8–9.5 months old; scale bar, 100 μm). One-way ANOVA with Tukey’s post hoc test; Student’s t-test for two-group comparisons; *P < 0.05, **P < 0.01, #P = 0.05; mean ± s.e.m.
Figure 2. Cord plasma improves neuronal function…
Figure 2. Cord plasma improves neuronal function within aged hippocampus
a , b , Population…
Figure 2. Cord plasma improves neuronal function within aged hippocampus a, b, Population spike amplitudes recorded in dentate gyrus granule cell layer by perforant path stimulation with representative traces before LTP induction (a/a′) and after LTP maintenance (b/b′) (n = 11–12 slices per group (see Source Data for range details) from n = 5 NSG mice per group; eight intravenous injections; 11.7 ± 0.2 months old). c, LTP maintenance-phase quantification. d, Days 1–4 escape latency and (e) fourth-day acquisition rate of cord-plasma-treated (n = 8) or vehicle-treated (n = 7) NSG mice in Barnes maze (eight intravenous injections; 12.9 ± 0.2 months old); T, trial. f, Contextual fear-conditioning freezing levels in separate cohort of cord-plasma-treated (n = 7) or vehicle-treated (n = 9) NSG mice (eight intravenous injections; 12.8 ± 0.2 months old). Two-way repeated-measures ANOVA with Bonferroni’s post hoc test for time × group comparisons; Student’s t-test for two-group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001; mean ± s.e.m.
Figure 3. Protein microarray analysis identifies putative…
Figure 3. Protein microarray analysis identifies putative pro-plasticity factors
a , Heat maps from unsupervised…
Figure 3. Protein microarray analysis identifies putative pro-plasticity factors a, Heat maps from unsupervised hierarchical cluster analysis of human (n = 15 cord, 19 young, 16 elderly subjects; |d-score| ≥ 1.30) or mouse (n = 8 mice per group; 3, 6, 12, 18, and 24 months of age; |d-score| ≥ 1.3) plasma protein changes analysed by significance analysis of microarray (SAM) for quantitative ageing correlations. Relative Z-scored values indicated in graded yellow (high) or blue (low). b, Number of c-Fos-positive cells in dentate gyrus from aged (16-month-old) WT mice treated four times every other day with 50 μg kg−1 (intraperitoneal) recombinant GAS6, CSF2, or TIMP2 or vehicle (n = 6 mice per group). c, ELISA-based plasma TIMP2 measurements from subjects in a. d, e, Representative TIMP2 immunoblot from equal volumes of 7- to 8-week-old or 20-month-old WT mouse plasma with corresponding Ponceau S stain (d) and quantification (e; n = 8 per group); see Supplementary Fig. 1 for uncropped image. f, Quantification of TIMP2+ cells with NeuN+ nuclei in dentate gyrus (hilus) by confocal microscopy of WT mice at indicated ages (n = 8, 8, 6, 8, 9 mice per group (left to right)). g, h, Brain uptake of 64Cu-labelled bovine serum albumin (BSA) (n = 4 mice per time point (n = 3 at 24 h)) and TIMP2 (n = 5 mice per time point) in WT mice (21-month-old) euthanized at indicated time points after injected (intraperitoneal) tracer dose (ID; g) and as a proportion of tracer remaining in blood (h). i, c-Fos-positive cell counts within dentate gyrus from WT mice in normal housing (NH) or enriched housing (EH) injected four times with vehicle or TIMP2 (n = 8, 8, 8, 7 per group (left to right); 20-month-old; *versus vehicle/normal housing; ‡versus vehicle/enriched housing; † versus TIMP2/normal housing). One-way ANOVA with Tukey’s post hoc test (Dunnett’s in b); two-way ANOVA with Bonferroni’s post hoc test for group × time comparisons; Student’s t-test for two-group comparisons; one, two, or four symbols denote P < 0.05, 0.01, 0.0001, respectively; mean ± s.e.m. Mouse schematic adapted from ref. .
Figure 4. Systemic TIMP2 treatment improves neuronal…
Figure 4. Systemic TIMP2 treatment improves neuronal function within aged hippocampus and is critical for…
Figure 4. Systemic TIMP2 treatment improves neuronal function within aged hippocampus and is critical for normal spatial memory and cognitive effects of cord plasma a–d, Barnes maze escape latency for all trial days (a), fourth-day acquisition rate (b), freezing levels during contextual fear-conditioning (c), and nesting behaviour within 24 h of providing intact nestlet (d) for aged WT mice given eight systemic vehicle or TIMP2 injections every other day (n = 15 per group; 20-month-old). e, f, Population spike amplitudes recorded in dentate gyrus granule cell layer following perforant path stimulation (e) and maintenance-phase LTP quantification (f; n = 10 slices per group from n = 5 mice per group; eight intravenous injections of vehicle or TIMP2; 20-month-old). g, h, PSA recorded in dentate gyrus granule cell layer following perforant path stimulation in slices incubated with indicated ex vivo treatments (g) and maintenance-phase LTP quantification (h; n = 10 slices per group from n = 7, 4, 5 WT mice per group (left to right); 2-month-old mice). i, Discrimination for novel object displacement on day 2 for young WT mice treated every other day for 4 weeks with anti-TIMP2 IgG or control IgG (n = 15 per group; 2.5-month-old). j–l, Latency to escape hole in Barnes maze (j), day 4 acquisition rate (k), and contextual fear-conditioning freezing levels (l) in aged NSG mice (13.8 ± 0.1 months old) given eight intravenous injections of vehicle (n = 10), TIMP2-depleted cord plasma (n = 8), or IgG control-depleted cord plasma (n = 9) (in j, * and † indicate control-depleted cord plasma versus either vehicle or TIMP2-depleted cord plasma groups, respectively). One-way ANOVA with Tukey’s post hoc test for h, k, l; two-way repeated-measures ANOVA with Bonferroni’s post hoc test for a, j; Student’s t-test for two-group comparisons; χ2 analysis for d; two-way ANOVA with Tukey’s post hoc test for i; *P < 0.05, #P = 0.05, **P < 0.01, † † † or ***P < 0.001, ****P < 0.0001; mean ± s.e.m.